ES2909579T3 - Arginine as sole nitrogen source for C1-fixing microorganisms - Google Patents

Abstract

A method of culturing a C1-fixing microorganism, comprising flowing a gaseous substrate comprising one or more of CO, CO2 and H2 into a bioreactor containing a culture of a C1-fixing microorganism in a liquid nutrient medium; where arginine is provided to the crop as the sole source of nitrogen; further, wherein the C1-binding microorganism is a recombinant genetically modified microorganism comprising an arginine metabolism pathway; and further wherein the C1-binding microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei.

Description

DESCRIPTION

Arginine as sole nitrogen source for C1-fixing microorganisms

The present application claims the benefit of US Provisional Patent Application Nos. 62/262,886 (filed December 3, 2015) and 62/262,888 (filed December 3, 2015).

Background of the invention

Currently, about 10% of the world's demand for energy and basic chemicals is produced from renewable feedstocks, mainly cultivated sugars. However, increasing attention is being paid to the future use of non-food resources to meet climate goals. Gas fermentation offers a way to use a wide range of low cost and readily available C1 feedstocks such as industrial waste gases, syngas or reformed methane in chemicals and fuels. The Wood Ljungdahl pathway, one of the earliest biochemical pathways to emerge on Earth, is believed to allow acetogenic Clostridia to fix these C1 gases into acetyl-CoA. Clostridium autoethanogenum, in particular, offers a robust and flexible platform for gas fermentation and has been implemented on an industrial scale. Gas fermentation by C. autoethanogenum is highly selective, tolerates contaminants, resolves the refractivity of Fischer-Tropsch processes, and is economically viable even when supplied with small volume gas streams.

It is known that while acetogenics are capable of producing many useful short-chain chemicals, the production of longer-chain carbon molecules for use in biodiesel or jet fuels is beyond the metabolic capacity of acetogenic bacteria by themselves, due to the limitation of ATP. Fast et al. determined that while the Wood Ljungdahl pathway was the best-yielding pathway for acetate and ethanol production, butanol fermentation is an ATP-limited process, making the anaerobic production of butanol from Wood Ljungdahl's route is ineffective.

C. autoethanogenum naturally produces acetate, ethanol, 2,3-butanediol (2,3-BDO), and lactate. If energetic impediments can be overcome, synthetic biology holds promise for improving the product spectrum of C. autoethanogenum. Acetogenic bacteria are widespread in nature and play an important role in the global carbon cycle, but are considered to live at the thermodynamic limit of life.

The energy of the Wood-Ljungdahl pathway of anaerobic acetogens is just emerging, but unlike aerobic growth conditions or glycolysis of sugar-fermenting organisms, in the Wood-Ljungdahl pathway ATP is not gained by phosphorylation at substrate level, in fact, the activation of CO2 to formate actually requires an ATP molecule and a membrane gradient is required [WO 2013/180584].

ATP generation through substrate-level phosphorylation can be used as a driving force for product synthesis, especially in ATP-limited systems. In particular, acetogenic bacteria are known to live at the thermodynamic limit of life (Schuchmann, Nat Rev Microbiol, 12: 809-821, 2014). Thus, all acetogenic microorganisms isolated to date have been reported to produce acetate (Drake, Acetogenic Prokaryotes, in: The Prokaryotes, 3rd edition, pages 354-420, New York, NY, Springer, 2006; Liew et al . al., Insights into CO2 Fixation Pathway of Clostridium autoethanogenum by Targeted Mutagenesis. mBio, 2016, 7: e00427-16) as acetate production provides the microorganism with an option to directly generate ATP from substrate-level phosphorylation via via Pta (phosphotransacetylase) (Ec 2.3.1.8) and Ack (acetate kinase) (EC 2.7.2.1). The Pta-Ack system is highly specific for the conversion of acetyl-CoA to acetate and does not use other acyl-CoAs. Although mechanisms such as membrane gradients and electron bifurcation enzymes coupled to ion or proton translocation systems, for example the Rnf complex (Schuchmann, Nat Rev Microbiol, 12: 809-821, 2014), conserve ATP in these microorganisms, the direct generation of ATP remains essential for their survival. As a result, when heterologous pathways are introduced that do not allow ATP generation, acetate is produced as a byproduct (Schiel-Bengelsdorf, FEBS Lett, 586: 2191 2198, 2012).

Summary of the invention

The invention provides a method for culturing a C1-fixing microorganism, comprising, flowing a gaseous substrate comprising one or more of CO, CO2 and H2 into a bioreactor containing a culture of a C1-fixing microorganism in a liquid nutrient medium. ; where arginine is provided to the crop as the sole source of nitrogen; further, wherein the C1-binding microorganism is a recombinant genetically modified microorganism comprising an arginine metabolism pathway; and further wherein the C1-binding microorganism is Clostridium autoethanogenum, Clostridium Ijungdahlii or Clostridium ragsdalei. The colulrue can be fermented to produce at least one product. Arginine can be provided to the crop in excess of the requirements for biomass synthesis.

Also disclosed is a method of increasing the production of at least one ATP-intensive product, the method comprising; flowing a gaseous substrate containing C1 into a bioreactor containing a culture of one or more C1-fixing microorganisms in a liquid nutrient medium; and fermenting the culture to produce at least one product. Arginine may be provided to the culture in excess of the requirements for biomass synthesis and the C1-fixing microorganism may comprise a pathway of arginine metabolism.

In particular embodiments, the arginine metabolism pathway comprises at least one arginine deiminase pathway and/or one arginine decarboxylase pathway. The arginine deiminase pathway comprises one or more enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6), ornithine carbamoyltransferase (putrescine carbamoyltransferase) (EC 2.1.3.3), and a carbamate kinase (EC 2.7.2.2). The arginine decarboxylase pathway comprises one or more enzymes selected from the group consisting of arginine decarboxylase (EC 4.1.1.19), possible agmatine deiminase (EC 3.5.3.12), putrescine carbamoyltransferase (EC 2.1.3.6), and carbamate kinase (EC 2.7). .2.2).

In general, the amount of arginine provided to the culture of C1-fixing microorganisms exceeds the requirement for biomass synthesis. Arginine can be provided to the culture from about the cellular requirement to about 1000 times above the cellular requirement. Arginine can be added to the crop from 2 to 1000 or from 2 to 800 or from 2 to 500 or from 2 to 100 or from 2 to 50 or from 2 to 10 or from 50 to 1000 or from 50 to 800 or from 50 to 600 or from 50 to 500 or from 50 to 300 or from 50 to 200 or from 50 to 100 or from 100 to 1000 or from 100 to 800 or from 100 to 600 or from 100 to 500 or from 100 to 300 or from 100 to 200 times the cell requirement.

Arginine can be provided to the culture, such that the arginine concentration is maintained in an amount of at least 20 mg/l or at least 100 mg/l or at least 300 mg/l or at least 500 mg/l or at least 1 g/l or at at least 2 g/l or at least 3 g/l or at least 4 g/l or at least 5 g/l or at least 10 g/l or at least 20 g/l. The arginine concentration can be maintained between 20 mg/l and approximately 20 g/l or between 100 mg/l and 20 g/l or between 500 mg/l and 20 g/l or between 500 mg/l and 10 g/l or between 1 g/l and 10 g/l or between 5 g/l and 10 g/l or between 5 g/l and 20 g/l. Arginine can be provided to the culture such that the consumption of arginine by the culture is at least 20 mg arginine per gram dry cell weight or at least 100 mg arginine per gram dry cell weight or at least 1 gram arginine. arginine per gram of dry cell weight or at least 5 grams of arginine per gram of dry cell weight or at least 10 grams of arginine per gram of dry cell weight. Arginine can be provided to the culture such that the consumption of arginine by the culture is between 20 mg and 20 grams per gram of dry cell weight or between 100 mg and 20 grams per gram of dry cell weight or between 1 gram and 10 grams per gram dry cell weight.

The culture can consume at least 0.012 g of arginine to produce 1 g of biomass. The cellular requirement of arginine for biomass synthesis can be between 0.012 g per gram of biomass and about 24 g per gram of biomass. The arginine requirement for biomass synthesis can be from at least 0.012 g per gram of biomass or at least 0.024 g per gram of biomass, to about 0.048 g per gram of biomass or at least 0.120 g per gram of biomass or at least 0.24 g per gram of biomass or at least 0.48 g per gram of biomass or at least 1.2 g per gram of biomass or at least 2.4 g per gram of biomass or at least 4.8 g per gram of biomass or at least 12 g per gram of biomass.

In general, arginine is provided to the C1-fixing microorganism culture as a component of the liquid nutrient medium that is continuously fed to the bioreactor. The arginine can be supplied to the bioreactor independently of the liquid nutrient medium (for example, by metering or continuous feeding). Culture doubling time can be increased by at least 10% or at least 20% or at least 30% or at least 40% or at least 50% or at least 60% or at least 70 % when arginine is provided to the culture in excess of the cellular requirements of the microorganism, compared to a culture where arginine is not provided in excess of the cellular requirements of the culture.

In particular embodiments, the C1-binding microorganism comprises at least one arginine deiminase pathway or one arginine decarboxylase pathway.

The C1-binding microorganism may be an acetogenic carboxydotrophic microorganism. Examples of suitable C1-fixing microorganisms include Clostridium, Moorella, Oxobacter, Peptostreptococcus, Acetobacterium, Eubacterium, or Butyribacterium. The microorganism may be selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Clostridium ragsdalei, Clostridium coskatii, Butyribacterium methylkalotrophicum, Alecchitobacterium woodaculutii, , Eubacterium limosum, Moorella thermoacetica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii and Thermoanaerobacter kiuvi.

In particular embodiments, the C1-fixing microorganism is Clostridium autoethanogenum or Clostridium ljungdahlii. In a particular embodiment, the microorganism is Clostridium autoethanogenum. The bacterium may have identification characteristics of registration numbers DSM10061, DSM19630, or DSM23693. These bacteria have been deposited at the German Resource Center for Biological Material (DSMZ) whose address is DSMZ GmbH InhoffenstraBe, 7 B, D-38124.

In certain embodiments, the C1-binding microorganism is selected from the group consisting of Clostridium autoethanogenum, Clostridium Ijungdahlii, and Clostridium ragsdalei.

The at least one product can be any product made by natural or recombinant C1-fixing microorganisms. The at least one product can be selected from the group consisting of ethanol, 2,3-butanediol, 1,3-butanediol, lactate, succinate, methyl ethyl ketone (MEK), butyrate, 2-butanol, 1,2-propanediol (1 ,2-PDO), 1-Propanol, Isopropanol (IPA), Acetoin, Iso-Butanol, Isoprene, Farnesene, Bisabolene, Pinene, Limonene, Acetone, 3-Hydroxybutyrate, 2-Hydroxyisobutyric Acid (2-HIBA), Citramalate, Butadiene , polylactic acid, 1-butanol, 3-hydroxypropionate (3-HP), benzoate, fatty acid ethyl esters, fatty acids and isobutylene.

The concentration of arginine provided to the culture can be increased to increase the production of at least one ATP-intensive product. In general, the rate of production of the at least one ATP-intensive product is at least 1.5-fold greater than the rate of production of the at least one ATP-intensive product when excess arginine is not provided for the cellular requirements of the culture. The production rate of the at least one ATP-intensive product may be at least 2-fold higher or at least 3-fold higher or at least 4-fold higher or at least 5-fold higher or at least 10-fold higher.

ATP intensive products are generally defined as products that have a direct requirement for ATP in the metabolic pathway. Examples of ATP-intensive products (products that directly require ATP for synthesis that have an ATP-dependent reaction pathway) include, but are not limited to, products from the terpenoid/mevalonate pathway, including isoprene, farnesene, bisabolene and limonene, products from the fatty acid pathway, including fatty acids, fatty acid ethyl esters, or molecules such as 3-hydroxypropionate (3-HP) or isobutylene.

The concentration of arginine provided to the culture can be increased to increase the production of at least one product selected from a group of products that do not directly require ATP but also do not produce the same amount of ATP per acetyl-CoA as the acetate formation. The concentration of arginine provided to the culture can be increased to increase the production of at least one product selected from the group consisting of acetone, IPA, 3-HB, 2-HIBA, 1,3-BDO, 2,3-BDO, lactate, 1,2-PDO, 1-propanol, iso-butanol, butyrate, butanol, citramalate, succinate and MEK.

The culture may produce a reduced amount of acetate compared to a culture where excess arginine is not provided for cellular requirements. The culture can produce at least 10% less acetate or at least 20% less acetate or at least 30% less acetate or at least 40% less acetate or at least 50% less acetate or at least least 60% less acetate or at least 80% less acetate. No net acetate may be produced, less than 1 g/l acetate, less than 0.5 g/l acetate, less than 0.1 g/l acetate, less than 0.05 g/l acetate, or less than 0.01 g/l acetate.

A method for improving the efficiency of a fermentation is disclosed, the method comprising providing the culture with one or more gaseous substrates and arginine, and fermenting the culture such that the culture utilizes argin and the one or more gaseous substrates. The gaseous substrate is selected from the group consisting of CO, H2 and CO2. The arginine and the gaseous substrate can be taken up by the culture in a ratio of at least 2:1 or at least 1:1 or at least 1:2.

Also disclosed is a method for improving the viability of a fermentation process, the method comprising flowing a C1-containing gaseous substrate into a bioreactor containing a culture of at least one C1-fixing bacterium comprising at least one arginine deiminase pathway or an arginine decarboxylase pathway in a liquid nutrient medium; and fermenting the culture to produce at least one product. Arginine can be provided to the culture in excess of the cellular requirements of the culture. In certain embodiments, the arginine provided to the crop is catabolized by the arginine deiminase pathway to produce ammonium, which is used by the crop as a nitrogen source.

A method of growing a C1-fixing bacterium with arginine as the sole nitrogen source is disclosed. Arginine may be provided to the culture and ammonium may not be provided extrinsically to the culture.

The C1-fixing bacterium may be selected from the group consisting of Clostridium, Moorella, Eubacterium, Oxobacter, Sporomusa and Thermoanaerobacter. The bacterium may be selected from the group consisting of Acetobacterium woodii, Alkalibaculum bacchii, Blautia product, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostricalo ragsdale , Eubacterium limosum, Moorella thermaautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Thermoanaerobacter kiuvi. In preferred embodiments, the bacterium is selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.

A genome-engineered C1-binding bacterium comprising an optimized arginine deiminase pathway is disclosed. The engineered C1-binding bacterium may comprise one or more enzymes selected from the group consisting of: arginine deiminase (Ec 3.5.3.6), carbamoyltransferase (ornithine carbamoyltransferase, putrescine carbamoyltransferase) (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2 ), where each enzyme is an overexpressed endogenous enzyme, a mutated endogenous enzyme, or an exogenous enzyme. The C1-binding bacterium may be a Clostridium bacterium. In particular embodiments, the bacterium is Clostridium autoethanogenum.

A genome-engineered C1-binding bacterium is disclosed that has improved cellular metabolism of arginine compared to a parent microorganism, wherein the genome-engineered C1-binding bacterium comprises one or more genetic modifications that disrupt an arginine transporter. The genetic modification can be an inactivation or replacement of the arginine:ornithine transporter (CAETHG 3023-24). The bacterium may further comprise one or more enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6), ornithine carbamoyltransferase (putrescine carbamoyltransferase) (EC 2.1.3.3), a carbamate kinase (EC 2.7.2.2), ornithine racemase ( EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12), 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10), where each enzyme is an overexpressed endogenous enzyme, a mutated endogenous enzyme, or an exogenous enzyme.

A method for producing at least one product from a substrate is disclosed, the method comprising culturing a genome-engineered C1-fixing bacterium comprising one or more enzymes selected from the group consisting of: arginine deiminase (EC 3.5.3.6), carbamoyltransferase ( ornithine carbamoyltransferase, putrescine carbamoyltransferase) (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2), where each enzyme is an overexpressed endogenous enzyme, an endogenous enzyme mutated from an exogenous enzyme.

A method for improving the efficiency of arginine uptake in metabolism is disclosed, the method comprising culturing a genome-engineered C1-binding bacterium comprising one or more genetic modifications selected from the group consisting of (i) a switch mutation that disrupts a transporter of arginine; (ii) overexpression of one or more endogenous enzymes selected from the group consisting of arginine deiminase ^{(EC 3.5.3.6), ornithine carbamoyltransferase (putrescine carbamoyltransferase)} (Ec 2.1.3.3), carbamate kinase (EC 2.7.2.2), ornithine racemase ( EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12), and 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10); (iii) expression of one or more mutated endogenous enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6), ornithine carbamoyltransferase (EC 2.1.3.3), carbamate kinase (EC 2.7.2.2), ornithine racemase (EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12) and 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10) and (iv) expression of one or more exogenous enzymes selected from the group consisting of arginine deiminase (Ec 3.5.3.6), ornithine carbamoyltransferase (putrescine carbamoyltransferase) (EC 2.1.3.3), carbamate kinase (EC 2.7.2.2), ornithine racemase (EC 5.1.1.12 ), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12), and 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10).

A method for enhancing the efficiency of co-utilizing arginine with one or more gaseous substrates selected from the group consisting of CO, H2 and CO2 is disclosed, the method comprising culturing a genome-engineered C1-fixing bacterium comprising one or more genetic modifications, wherein the one or more genetic modifications are selected from the group consisting of (i) switch mutation of regulatory elements and (ii) replacement of operator binding sites or natural promoters with constitutive or synthetic promoters. In particular embodiments, the disruptive mutation is an inactivation of the argR arginine repressor, and the replacement is a replacement of an arginine deiminase pathway operon promoter with a constitutive or synthetic promoter.

Brief description of the drawings

Figure 1 is a graph showing the growth of C. autoethanogenum DSM10061 in 20 amino acid defined medium 5 g fructose/L.

Figure 2 is a graph showing amino acid consumption and production data for DSM10061 from C. autoethanogenum growing in 20AA medium. The marker indicates the OD at the time of sampling; the cysteine measurement for the first two samples was outside the calibration range.

Figure 3 is a graph showing the growth of C. autoethanogenum DSM10061 in 14AA and 8AA media along with a comparison to 20AA media. Error bars represent the standard deviation between duplicate cultures. 4* and 2* mean 4-fold and 2-fold higher AA concentrations compared to the 20AA medium, respectively.

Figure 4 is a graph showing the doubling time, tD, for C. autoethanogenum DSM10061 for 14AA and 8AA media compared to 20AA media. tD, doubling time; Error bars represent the standard deviation between duplicate cultures. 4* and 2* mean 4-fold and 2-fold higher AA concentrations compared to the 20AA medium, respectively.

Figure 5 is a graph showing the amino acid consumption and production data of C. autoethanogenum DSM10061 in 14 AA 4* medium. 4* means 4-fold higher AA concentrations in comparison with the mean of 20AA. The marker indicates the OD at the time of sampling.

Figure 6 is a graph showing the amino acid consumption and production data of DSM10061 from C. autoethanogenum in 2*8AA medium. 2* means 2-fold higher AA concentrations compared to the 20AA medium. The marker indicates the OD at the time of sampling; the cysteine measurement for the first sample was outside the calibration range.

Figure 7 is a graph showing the growth data of C. autoethanogenum DSM10061 in 12AA and 4AA media. Error bars represent the standard deviation between duplicate cultures.

Figure 8 is a graph showing amino acid consumption and production data for C. autoethanogenum DSM10061 in 12 AA medium. The marker indicates the OD at the time of sampling.

Figure 9 is a graph showing amino acid consumption and production data for C. autoethanogenum DSM10061 in 4AA medium. The marker indicates the OD at the time of sampling; the pM unit is not applicable to the maximum of 8.3 min.

Figure 10 is a graph showing growth data of C. autoethanogenum DSM10061 in 4AA medium using BugLab. Error units, raw data input. The specific growth rate 0.34 h-1 is tD~2 h. Figure 11 is a graph showing the growth data of C. autoethanogenum DSM10061 in PETC-MES media without YE with 5 g arginine/L 5 g fructose/L.

Figure 12 is a graph showing acetate production data for C. autoethanogenum DSM10061 in PETC-MES medium without YE with 5 g arginine/L 5 g fructose/L.

Figure 13 is a graph showing amino acid consumption and production data for C. autoethanogenum DSM10061 at 5 g arginine/l 5 g fructose/l. The marker indicates the OD at the time of sampling; the pM unit is not applicable to the maximum of 8.3 min.

Figure 14 is a graph showing the growth data of C. autoethanogenum DSM10061 in PETC-MES medium without YE with 5 g arginine/L 5 g fructose/L using BugLab. Error units, raw data input. The specific growth rate 0.21 h-1 is tD~3 h.

Figure 15 is a graph showing the difference in growth curve and doubling times of arginine and yeast extract in fructose bioreactor experiments.

Figure 16 is a graph showing autotrophic growth of C. autoethanogenum DSM23693 with (A) and without (•) arginine supplements.

Figure 17 is a graph showing autotrophic growth of C. autoethanogenum DSM23693 with (A) and without (•) arginine supplements, as well as growth on arginine alone in the absence of CO/CO2/H2 gas (□). Figure 18 is a log graph of autotrophic growth of C. autoethanogenum DSM23693 with (A) and without (•) arginine supplements.

Figure 19 is a graph showing acetate production during autotrophic growth of C. autoethanogenum DSM23693 with (A) and without (•) arginine supplements.

Figure 20 is a schematic representation of the arginine deiminase pathway.

Figure 21 is a schematic representation of L-arginine degradation in an arginine deiminase pathway.

Figure 22A and Figure 22B are pie charts showing carbon flow to products.

Figure 23 shows the enzymes required for ornithine uptake in Clostridium sticklandii.

Figure 24 is a graph showing the distribution of ATP production during heterotrophic growth of C. autoethanogenum in 4AA or ARG medium with fructose.

Detailed description of the invention

Definitions and background

The term "in excess of cellular requirements" refers to providing a component to the microorganism that is greater than the amount of the component needed by the microorganism for biomass synthesis.

The term "ATP-intensive product" refers to a metabolite whose synthesis requires the input of ATP (energy) at least in one step of its synthetic pathway (eg, which has an ATP-dependent reaction in the pathway).

The term "specific growth rate" refers to the growth rate of cell biomass per cell biomass per hour.

The term "doubling time" refers to the time in hours it takes for cell biomass to double. The term "arginine metabolism pathway" refers generally to any pathway involved in arginine metabolism. The arginine metabolism pathway typically comprises at least one arginine deiminase pathway and one arginine decarboxylase pathway.

The expression "genetic modification" or "genomanipulation" generally refers to the manipulation of the genome or the nucleic acids of a microorganism. Similarly, the term "genetically engineered" refers to a microorganism comprising a manipulated genome or nucleic acids. Genetic modification methods include, for example, heterologous gene expression, gene or promoter insertion or deletion, nucleic acid mutation, altered gene expression or inactivation, enzyme engineering, genetic evolution directed, knowledge-based design, random mutagenesis methods, gene rearrangement and codon optimization.

"Recombinant" indicates that a nucleic acid, protein, or microorganism is the product of genetic modification, genome engineering, or recombination. In general, the term "recombinant" refers to a nucleic acid, protein, or microorganism that contains or is encoded by genetic material from multiple sources, such as two or more different strains or species of microorganisms. As used herein, the term "recombinant" may also be used to describe a microorganism that comprises a mutated nucleic acid or protein, including a mutated form of an endogenous nucleic acid or protein.

"Endogenous" refers to a nucleic acid or protein that is present or expressed in the wild-type or precursor microorganism from which the microorganism is derived. For example, an endogenous gene is a gene that is naturally present in the wild-type or precursor microorganism from which the microorganism is derived. Expression of an endogenous gene may be controlled by an exogenous regulatory element, such as an exogenous promoter. "Exogenous" refers to a nucleic acid or protein that is not present in the wild-type or precursor microorganism from which the microorganism is derived. An exogenous gene or enzyme may originate from a heterologous (ie, different) strain or species and be introduced or expressed in the microorganism. An exogenous gene or enzyme may be artificially or recombinantly created and introduced or expressed in the microorganism. Exogenous nucleic acids can be adapted to integrate into the genome of the microorganism to remain in an extrachromosomal state in the microorganism, for example, in a plasmid.

"Enzyme activity," or simply "activity," refers broadly to enzyme activity, including, but not limited to, the activity of an enzyme, the amount of an enzyme, or the availability of an enzyme to catalyze a reaction. Accordingly, "increasing" enzyme activity includes increasing the activity of an enzyme, increasing the amount of an enzyme, or increasing the availability of an enzyme to catalyze a reaction. Similarly, "decreasing" enzyme activity includes decreasing the activity of an enzyme, decreasing the amount of an enzyme, or decreasing the availability of an enzyme to catalyze a reaction. "Mutated" refers to a nucleic acid or protein that has been modified in the microorganism as compared to the wild-type or precursor microorganism from which the microorganism was derived. The mutation can be a deletion, insertion, or substitution in a gene that encodes an enzyme. The mutation can be a deletion, insertion, or substitution of one or more amino acids in an enzyme.

In particular, a "switching mutation" is a mutation that reduces or eliminates (ie, "disrupts") the expression or activity of a gene or enzyme. The switch mutation can partially inactivate, totally inactivate, or eliminate the gene or enzyme. The disruptive mutation may be an inactivating (KO) mutation. The disruptive mutation can be any mutation that reduces, prevents, or blocks the biosynthesis of a product generated by an enzyme. Switching mutation may include, for example, a mutation in a gene that encodes an enzyme, a mutation in a genetic regulatory element involved in the expression of a gene that encodes an enzyme, the introduction of a nucleic acid that produces a protein that reduces or inhibits the activity of an enzyme or the introduction of a nucleic acid (eg, antisense RNA, siRNA, CRISPR) or protein that inhibits the expression of an enzyme. The switch mutation can be introduced using any method known in the art.

Introduction of a switch mutation results in a microorganism that does not produce any target product or that produces substantially no target product or that produces a reduced amount of target product compared to the parent microorganism from which the microorganism is derived. For example, the microorganism may not produce any target product or at least about 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60% , 70%, 80%, 90% or 95% less target product than the parent microorganism. For example, the microorganism may produce less than about 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g/L of target product.

"Codon optimization" refers to the mutation of a nucleic acid, such as a gene, for optimized or enhanced translation of the nucleic acid in a particular strain or species. Codon optimization can result in faster translation speeds or higher translation accuracy. The genes may be codon optimized for expression in Clostridium, in particular Clostridium autoethanogenum, Clostridium Ijungdahlii or Clostridium ragsdalei. The genes may be codon-optimized for expression in Clostridium autoethanogenum LZ1561, which is deposited under DSMZ accession number DSM23693.

"Overexpressed" refers to an increase in the expression of a nucleic acid or protein in the microorganism as compared to the wild-type or precursor microorganism from which the microorganism is derived. Overexpression can be achieved by any means known in the art, including modifying gene copy number, gene transcription rate, gene translation rate, or enzymatic degradation rate.

The term "variants" includes nucleic acids and proteins whose sequence varies from the sequence of an acid reference nucleic acid and protein, such as a sequence of a reference nucleic acid and protein disclosed in the prior art or exemplified herein. The invention can be practiced using variant nucleic acids or proteins that perform substantially the same function as the reference nucleic acid or protein. For example, a variant protein can perform substantially the same function or catalyze substantially the same reaction as a reference protein. A variant gene may encode the same or substantially the same protein as a reference gene. A variant promoter may have substantially the same ability to promote expression of one or more genes as a reference promoter.

Such nucleic acids or proteins may be referred to herein as "functionally equivalent variants." By way of example, functionally equivalent variants of a nucleic acid can include allelic variants, fragments of a gene, mutated genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. These include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii, or Clostridium Ijungdahlii, details of which are publicly available on websites such as Genbank or NCBI. Functionally equivalent variants also include nucleic acids whose sequence varies as a result of codon optimization for a particular microorganism. A functionally equivalent variant of a nucleic acid will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or more sequence identity to the nucleic acid. (percent homology) with the reference nucleic acid. A functionally equivalent variant of a protein will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or more amino acid identity (percent homology). ) with the reference protein. The functional equivalence of a variant nucleic acid or protein can be assessed using any method known in the art.

Nucleic acids can be administered to a microorganism using any method known in the art. For example, the nucleic acids can be administered as naked nucleic acids or can be formulated with one or more agents, such as liposomes. Nucleic acids can be DNA, RNA, cDNA, or combinations thereof, as appropriate. Restriction inhibitors can be used. Additional vectors may include plasmids, viruses, bacteriophages, cosmids, and artificial chromosomes. Nucleic acids can be delivered to the microorganism using a plasmid. By way of example, transformation (including transduction or transfection) can be achieved by electroporation, ultrasound, polyethylene glycol-mediated transformation, chemical or natural competition, protoplast transformation, prophage induction, or conjugation. When an active restriction enzyme system is present, it may be necessary to methylate a nucleic acid prior to introduction of the nucleic acid into a microorganism.

In addition, nucleic acids can be designed to comprise a regulatory element, such as a promoter, to enhance or otherwise control expression of a particular nucleic acid. The promoter can be a constitutive promoter or an inducible promoter. Ideally, the promoter is a Wood-Ljungdahl pathway promoter, a ferredoxin promoter, a pyruvate:ferredoxin oxidoreductase promoter, an Rnf complex operon promoter, an ATP synthase operon promoter, or an operon promoter. of phosphotransacetylase/acetate kinase.

A "microorganism" is a microscopic organism, especially a bacterium, archaea, virus, or fungus. Typically, the microorganism is a bacterium. As used herein, reference to "microorganism" should be taken to encompass "bacteria".

A "precursor microorganism" is a microorganism used to generate a microorganism. The parent microorganism may be a naturally occurring microorganism (ie, a wild-type microorganism) or a microorganism that has been previously modified (ie, a mutant or recombinant microorganism). The microorganism can be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parent microorganism. Similarly, the microorganism can be modified to contain one or more genes that were not contained in the parent microorganism. The microorganism may also be modified not to express or to express lesser amounts of one or more enzymes than were expressed in the parent microorganism. In one embodiment, the parent microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the parent organism is Clostridium autoethanogenum LZ1561, which was deposited on June 7, 2010 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), located at Inhoffenstrap 7B, D-38124 Braunschwieg, Germany, on June 7. June June 2010 under the terms of the Budapest Treaty and assigned registration number DSM23693.

The term "derived from" indicates that a nucleic acid, protein, or microorganism is modified or adapted from a different nucleic acid, protein, or microorganism (for example, a precursor or wild-type), to produce a new nucleic acid, protein, or protein. or microorganism. Such modifications or adaptations generally include insertion, deletion, mutation, or substitution of nucleic acids or genes. Generally, the microorganism originates from a precursor microorganism. In one embodiment, the microorganism is from Clostridium autoethanogenum, Clostridium Ijungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the microorganism is from Clostridium autoethanogenum LZ1561, which is deposited under DSMZ accession number DSM23693.

The microorganism can be further classified based on functional characteristics. For example, the microorganism may be or be derived from a C1-fixing microorganism, an anaerobe, an acetogen, an ethanologen, a carboxydotroph, and/or a methanotroph. Table 1 provides a representative list of microorganisms and identifies their functional characteristics.

1 Acetobacterium woodi can produce ethanol from fructose, but not from gas.

2 Whether Clostridium magnum can grow in CO has not been investigated.

3 A strain of Moorella thermoacetica, Moorella sp. HUC22-1, has been reported to produce ethanol from gas.

4 Whether Sporomusa ovata can grow in CO has not been investigated.

5 Whether Sporomusa silvacetica can grow in CO has not been investigated.

6 Whether Sporomusa sphaeroides can grow in CO has not been investigated.

"C1" refers to a one-carbon molecule, eg, CO, CO2, CH4, or CH3OH. "C1-oxygenated" refers to a one-carbon molecule that also comprises at least one oxygen atom, eg, CO, CO2, or CH3OH. "C1 carbon source" refers to a carbon molecule that serves as a partial or sole carbon source for the microorganism. For example, a C1 carbon source may comprise one or more of CO, CO2, CH4, CH3OH, or CH2O2. Preferably, the C1 carbon source comprises one or both of CO and CO2. A "C1-fixing microorganism" is a microorganism that has the ability to produce one or more products from a C1 carbon source. Typically, the organism is a C1-fixing bacterium. The organism may be derived from a C1-binding organism identified in Table 1.

An "anaerobe" is a microorganism that does not need oxygen for growth. An anaerobe can react negatively or even die if oxygen is present above a certain threshold. Normally, the microorganism is an anaerobe. The microorganism may originate from an anaerobe identified in Table 1. An "acetogen" is an obligate anaerobic bacterium that uses the Wood-Ljungdahl pathway as its main mechanism for energy conservation and for the synthesis of acetyl-CoA and derivative products. of acetyl-CoA, such as acetate (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008). Acetyl-CoA pathway is used by acetogens as a (1) mechanism for reductive synthesis of acetyl-CoA from CO2, (2) terminal process of electron acceptance and energy conservation, (3) mechanism for fixation (assimilation) of CO2 in cellular carbon synthesis (Drake, Acetogenic Prokaryotes, in: The Prokaryotes, 3rd edition, p. 354, New York, NY, 2006). All naturally occurring acetogens are C1-fixing, anaerobic, autotrophic, and non-methanotrophic. Typically, the microorganism is an acetogen. The microorganism may come from an acetogen identified in Table 1.

An "ethanologen" is a microorganism that produces or is capable of producing ethanol. Typically, the microorganism is an ethanologen. The microorganism may be derived from an ethanologen identified in Table 1. An "autotroph" is a microorganism capable of growth in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources, such as CO and/or CO2. Normally, the microorganism is an autotroph. The microorganism may come from an autotroph identified in Table 1.

A "carboxydotroph" is a microorganism capable of using CO as its sole carbon source. Normally, the microorganism is a carboxydotroph. The microorganism may come from a carboxydotroph identified in Table 1.

A "methanotroph" is a microorganism capable of using methane as its sole source of carbon and energy. The microorganism may originate from a methanotroph.

More broadly, the microorganism may be from any genus or species identified in Table 1. In a preferred embodiment, the microorganism may be from the Clostridia group comprising Clostridium autoethanogenum, Clostridium Ijungdahlii, and Clostridium ragsdalei species. These species were first reported and characterized by Abrini, Arch Microbiol, 161:345-351, 1994 ( Clostridium autoethanogenum), Tanner, Int J System Bacteriol, 43:232-236, 1993 ( Clostridium ljungdahlii) , and Huhnke, WO 2008/028055 ( Clostridium ragsdalei).

These three species have many similarities. In particular, these species are all C1-fixing, anaerobic, acetogenic, ethanologenic, and carboxydotrophic members of the genus Clostridium . These species have similar genotypes and phenotypes and modes of energy conservation and fermentative metabolism. Furthermore, these species are grouped in clostridial rRNA homology group I with 16S rRNA DNA that is more than 99% identical, have a G+C content in DNA of approximately 22 to 30 mol%, are gram-positive, have similar morphology and size (log-growing cells between 0.5 to 0.7 * 3 to 5 µm), are mesophilic (grow optimally at 30 to 37°C), have pH ranges similar from about 4 to 7.5 (with a pH optimum of about 5.5 to 6), lack cytochromes and conserve energy through an Rnf complex. Also, the reduction of carboxylic acids in their corresponding alcohols has been demonstrated in these species (Perez, Biotechnol Bioeng, 110:1066-1077, 2012). Notably, these species also show strong autotrophic growth in CO-containing gases, produce ethanol and acetate (or acetic acid) as major fermentation products, and produce small amounts of 2,3-butanediol and lactic acid under certain conditions.

However, these three species also have several differences. These species were isolated from different sources: Clostridium autoethanogenum from rabbit gut, Clostridium ljungdahlii from chicken coop waste, and Clostridium ragsdalei from freshwater sediments. These species differ in their utilization of various sugars (eg, rhamnose, arabinose), acids (eg, gluconate, citrate), amino acids (eg, arginine, histidine), and other substrates (eg, betaine, butanol). Furthermore, these species differ in auxotrophy to certain vitamins (eg, thiamin, biotin). These species have differences in the nucleic acid and amino acid sequences of the genes and proteins of the Wood-Ljungdahl pathway, although the general organization and number of these genes and proteins have been found to be the same in all species. (Kopke, Curr Opin Biotechnol, 22: 320-325, 2011).

Therefore, in summary, many of the characteristics of Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei are not specific to that species, but rather are general characteristics of this group of C1-fixing, anaerobic, acetogenic, ethanologenic Clostridium members. and carboxydotrophic. However, since these species are, in fact, distinct, genetic modification or manipulation of one of these species may not have an identical effect on another of these species. For example, differences in product growth, yield or production may be observed.

The microorganism of the invention can also come from an isolate or mutant of Clostridium autoethanogenum, Clostridium Ijungdahlii or Clostridium ragsdalei. Clostridium autoethanogenum isolates and mutants include JA1-1 (DSM10061) (Abrini, Arch Microbiol, 161: 345-351, 1994), LBS1560 (DSM19630) (WO 2009/064200), and LZ1561 (DSM23693). Isolates and mutants of Clostridium ljungdahlii include ATCC 49587 (Tanner, Int J Syst Bacteriol, 43:232-236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (US 5,593,886), C-01 (ATCC 55988) (US 6,368,819), O-52 (ATCC 55989) (US 6,368,819) and OTA-1 (Tirado-Acevedo, "Production of bioethanol from synthesis gas using Clostridium ljungdahlii', PhD thesis , North Carolina State University, 2010) Isolates and mutants of Clostridium ragsdalei include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).

"Substrate" refers to a carbon and/or energy source for the microorganism. Typically, the substrate is gaseous and comprises a C1 carbon source, eg CO, CO2 and/or CH4. Preferably, the substrate comprises a C1 carbon source of CO or COCO2. The substrate may further comprise other non-carbon components, such as H2, N2 or electrons.

The substrate generally comprises at least some amount of CO, such as about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mole % CO. The substrate may comprise a range of CO, such as about 20 to 80, 30 to 70, or 40 to 60 mole % CO. Preferably, the substrate comprises about 40 to 70 mol% CO (eg, steel mill or blast furnace gas), about 20 to 30 mol% CO (eg, basic oxygen furnace gas), or about 15 to 45 mol% CO (eg syngas). The substrate may comprise a relatively low amount of CO, such as about 1 to 10 or 1 to 20 mole % CO. The microorganism normally converts at least a portion of the CO in the substrate to a product. The substrate may comprise no or substantially no (<1 mol%) CO.

The substrate may comprise some amounts of H2. For example, the substrate may comprise about 1, 2, 5, 10, 15, 20 or 30 mole % H2. The substrate may comprise a relatively high amount of H2, such as about 60, 70, 80 or 90 mole % H2. The substrate may comprise no or substantially no (<1 mol%) H2.

The substrate may comprise some amounts of CO2. For example, the substrate may comprise about 1 to 80 or 1 to 30 mole % CO2. The substrate may comprise less than about 20, 15, 10 or 5 mole % CO2. The substrate may comprise no or substantially no (<1 mol%) CO2.

Although the substrate is normally gaseous, the substrate can also be provided in alternative forms. For example, the substrate can be dissolved in a liquid saturated with a gas containing CO using a microbubble dispersion generator. By way of further example, the substrate may be adsorbed onto a solid support. The substrate and/or carbon source C1 may be a waste gas obtained as a by-product of an industrial process or from some other source, such as automobile exhaust or biomass gasification. The industrial process can be selected from the group consisting of the manufacture of ferrous metal products, such as the manufacture of a steel mill, the manufacture of non-ferrous products, petroleum refining processes, coal gasification, electric power production, black production. coal production, ammonia production, methanol production and coke manufacturing. In the present case, the substrate and/or the C1 carbon source can be captured from the industrial process before being emitted to the atmosphere, using any convenient method. The substrate and/or carbon source C1 may be syngas, such as syngas obtained by gasification of coal or refinery residues, gasification of biomass or lignocellulosic material, or reforming of natural gas. Synthesis gas can be obtained from the gasification of urban solid waste or industrial solid waste.

The composition of the substrate can have a significant impact on the efficiency and/or cost of the reaction. For example, the presence of oxygen (O2) can reduce the efficiency of an anaerobic fermentation process. Depending on the composition of the substrate, it may be desirable to treat, wash, or filter the substrate to remove any undesirable impurities, such as toxins, unwanted components, or dust particles, and/or to increase the concentration of desirable components.

The microorganism can be cultured to produce one or more products. For example, Clostridium autoethanogenum produces or can be engineered to produce ethanol (WO 2007/117157), acetate (WO 2007/117157), butanol (WO 2008/115080 and WO 2012/053905), butyrate (WO 2008/ 115080), 2,3-butanediol (WO 2009/151342), lactate (WO 2011/112103), butene (WO 2012/024522), butadiene (WO 2012/024522), methyl ethyl ketone (2-butanone) ( WO 2012/024522 and WO 2013/185123), ethylene (WO 2012/026833), acetone (WO 2012/115527), isopropanol (WO 2012/115527), lipids (WO 2013/036147), 3 -hydroxypropionate (3-hP) (WO 2013/180581), isoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol (WO 2013/185123), 1,2-propanediol (WO document 2014/0369152) and 1-propanol (WO 2014/0369152). In addition to one or more target products, the microorganism can also produce ethanol, acetate, and/or 2,3-butanediol. The microbial biomass itself can be considered a product.

A "natural product" is a product generated by a non-genetically modified microorganism. For example, ethanol, acetate, and 2,3-butanediol are natural products of Clostridium autoethanogenum, Clostridium Ijungdahlii, and Clostridium ragsdalei. An "unnatural product" is a product that is produced by a genetically modified microorganism, but is not produced by the non-genetically modified microorganism from which the genetically modified microorganism originated.

"Selectivity" refers to the ratio of the generation of a target product to the generation of all fermentation products generated by a microorganism. The microorganism can be engineered to generate products at a certain selectivity or at a minimal selectivity. A target product may account for at least about 5%, 10%, 15%, 20%, 30%, 50%, or 75% of all fermentation products generated by the microorganism. The target product may account for at least 10% of all fermentation products generated by the microorganism, such that the microorganism has a selectivity for the target product of at least 10%. The target product can account for at least 30% of all the fermentation products generated by the microorganism, so that the microorganism has a selectivity for the target product of at least 30%.

"Efficiency-increasing," "increased efficacy," and the like include, but are not limited to, increased specific growth rate, rate or volume of product generation, volume of product per volume of substrate consumed, or product selectivity. Efficacy can be measured relative to the performance of the precursor microorganism from which the microorganism is derived.

The terms "productivity" or "production rate" are the volumetric productivity of a product. In continuous systems, volumetric productivity is calculated as the ratio of the steady-state concentration of the product to the liquid retention time. In batch systems, volumetric productivity is calculated as the concentration and the time required to produce that concentration in a batch system. Volumetric productivity is expressed as g/l/day. Normally, the cultivation is carried out in a bioreactor. The term "bioreactor" includes a culture/fermentation device consisting of one or more vessels, towers, or piping structures, such as a continuous stirred tank reactor (CSTR), a immobilized cell reactor (ICR), trickle bed reactor (TBR), bubble column, gas-lift fermenter, static mixer, or other suitable vessel or device for gas-liquid contact. The bioreactor may comprise a first growth reactor and a second culture/fermentation reactor. The substrate can be provided to one or both of these reactors. As used herein, the terms "culture" and "fermentation" are used interchangeably. These terms encompass both the growth phase and the product biosynthesis phase of the cultivation/fermentation process.

The culture is generally maintained in an aqueous culture medium containing sufficient nutrients, vitamins, and/or minerals to allow growth of the microorganism. Preferably, the aqueous culture medium is an anaerobic microbial growth medium, such as minimal anaerobic microbial growth medium. Suitable media are well known in the art.

It is desirable that the cultivation/fermentation be carried out under conditions appropriate for the production of the target product. Normally, cultivation/fermentation is carried out under anaerobic conditions. The reaction conditions to be considered include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, pH of the media, redox potential of the media, stirring speed (in case of using a mixed flow stirred tank reactor). continuous), inoculum level, maximum gaseous substrate concentrations to ensure that gas in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition. In particular, the rate of introduction of the substrate can be controlled to ensure that the gas concentration in the liquid phase does not become limiting, since the products can be consumed by the culture under gas-limited conditions.

Operation of a bioreactor at elevated pressures allows for an increased rate of gas mass transfer from the gas phase to the liquid phase. Accordingly, it is generally preferable to carry out cultivation/fermentation at pressures greater than atmospheric pressure. Also, since a given gas conversion rate is, in part, a function of substrate retention time and retention time dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the substrate. required bioreactor and, consequently, the capital expenditure of the culture/fermentation equipment. This, in turn, means that retention time, defined as the liquid volume in the bioreactor divided by the feed gas flow rate, can be reduced when the bioreactors are kept at elevated pressure, rather than atmospheric pressure. Optimum reaction conditions will depend in part on the particular microorganism used. However, in general, it is preferable to operate the fermentation at pressures greater than atmospheric pressure. Also, since a given gas conversion rate is in part a function of the substrate retention time and achieving a time As the desired retention rate, in turn, dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the required volume of the bioreactor and thus the capital cost of the fermentation equipment.

The target products can be separated or purified from a fermentation broth using any method or combination of methods known in the art, including, for example, fractional distillation, evaporation, pervaporation, gas stripping, phase separation, and extractive fermentation, including , for example, liquid-liquid extraction. Target products can be recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering one or more target products from the broth. The alcohols and/or acetone can be recovered, for example, by distillation. The acids can be recovered, for example, by adsorption on activated carbon. The separated microbial cells are preferentially returned to the bioreactor. Cell-free permeate remaining after target products have been removed is preferably also returned to the bioreactor. Additional nutrients (such as B vitamins) can be added to the cell-free permeate to replenish the nutrient medium before it is returned to the bioreactor.

The use of the term "acid", "acids" and the like when referring to the addition of an "acid" to a culture or bioreactor according to the invention should be taken in a broad sense, including any monocarboxylic and dicarboxylic acid. Reference to the addition or production of equivalent salts should be understood as referring to the acid, or a mixture, therefore. For example, reference to the term acetate should include acetic acid and vice versa. The ratio of molecular acid to carboxylate in the fermentation broth depends on the pH of the system. Examples of acids include acetic acid, propionic acid, n-butyric acid, n-pentanoic acid, n-hexanoic acid, and benzoic acid.

Methods for improving fermentation efficiency are disclosed. The inventors have discovered that the addition of specific amino acids in excess of cellular requirements to a microbial culture has a profound effect on the growth of the culture. Furthermore, the inventors have identified that this excess addition of amino acids allows for increased production of fermentation products, especially fermentation products with high energy/ATP demands.

The present disclosure provides methods for improving fermentation efficiency. The inventors have discovered that the addition of arginine in excess of cellular requirements to a microbial culture has a profound effect on the growth of the culture. Furthermore, the inventors have identified that this excess addition of arginine allows for increased production of fermentation products, especially fermentation products with high energy/ATP demands. Without wishing to be bound by theory, it is believed that the growth-enhancing effect of arginine results from the production of ATP during catabolism of arginine by the microorganism. Arginine is catabolized through an arginine deiminase (ADI) pathway or an arginine decarboxylase pathway.

The catabolism of arginine through arginine deiminase occurs by the following mechanism:

The catabolism of arginine through the ADI pathway results in the production of ammonia, CO2, and ATP. The catabolism of arginine through the arginine decarboxylase pathway occurs by the following mechanism:

Arginine decarboxylase Possible agmatine deaminase Putrescine carbamoyl

SicA , ,, u ,A» transferesa ^L il

Carban mato

Carban killed

2.7.22 Arp ciñ na: sa Ammonio J

2.7.2 2Arp

The production of ATP resulting from the catabolism of arginine allows a greater growth profile of the microorganism.

Without wishing to be bound by theory, the inventors believe that while arginine catabolism provides the microorganism's ATP requirement for growth, arginine is not used as a carbon source by the microorganism, rather, the carbon source used is the carbon component in the gas that contains C1. Processes for the microbial fermentation of C1-containing gaseous substrates to generate products such as ethanol and acetate are well known in the art. Such processes provide a means of producing commercially useful fuels from industrial waste gases comprising C1 carbon sources. These processes generally involve feeding a gaseous substrate comprising, for example, CO to a bioreactor comprising a culture of at least one acetogenic carboxydotrophic microorganism in a liquid nutrient medium. The gaseous substrate is anaerobically fermented to produce alcohols, acids, and mixtures thereof. The liquid nutrient medium used in the bioreactor normally contains various nutrients that support the growth of at least one acetogenic carboxydotrophic microorganism and are used in metabolic pathways of the one or more microorganisms to produce alcohols. Examples of such nutrients include MgCl, CaCl, KCl, H3PO4, Fe, Ni, Zn, Mn, B, W, Se, etc.

It is also known that Clostridium strains can be genetically modified to allow the production of a number of other useful products, including succinate, methyl ethyl ketone (MEK), 2-butanol, propanediol, 2-propanol, isopropanol, isoprene, acetoin, iso-butanol, citramalate, butadiene and polylactic acid, acetone, butanol, isobutylene, 3-hydroxypropionate (3HP) and fatty acids.

Surprisingly, the inventors have found that increasing the concentration of arginine provided to the microbial culture increased the growth profile of the microorganism and increases the amount of ATP-intensive products that the culture can produce.

One example involves providing a liquid nutrient medium with arginine, where the amount of arginine provided by the liquid nutrient medium exceeds the cellular requirements of the C1-fixing microorganism. Providing arginine in excess of the cellular requirements of the C1-fixing microorganism has the effect of increasing the specific growth rate of the microbial culture. The specific growth rate of the C1-binding microorganism can be increased by at least 10% or at least 20% or at least 30% or at least 40% or at least 50% or at least 60% or by less than 70% compared to the microorganism without excess arginine.

The cellular requirement for arginine by a microorganism can be estimated by determining the amino acid composition of the biomass after biomass hydrolysis and amino acid analysis. It has been shown that increasing the concentration of arginine in liquid nutrient media from matching the cellular requirement to 1000 times (or more) above the requirement for biomass synthesis, decreased the doubling time of the microorganism. In certain cases, the doubling time of the culture provided with excess arginine decreased to 3.5 h compared to a doubling time of 7.3 h in a culture without excess arginine. Without wishing to be bound by theory, the inventors also believe that increasing the arginine concentration to 2 to 80 times (or more) above the cellular requirement increases the productivity rate of ATP intensive fermentation products. Arginine can be provided to the culture from 2 to 1000 or from 2 to 800 or from 2 to 500 or from 2 to 100 or from 2 to 50 or from 2 to 10 or from 50 to 1000 or from 50 to 800 or from 50 to 600 , or from 50 to 500 or from 50 to 300 or from 50 to 200 or from 50 to 100 or from 100 to 1000 or from 100 to 800 or from 100 to 600 or from 100 to 500 or from 100 to 300 or from 100 to 200 times the requirement for biomass synthesis.

In terms of actual concentration, this concentration may be one where the concentration of arginine in the liquid nutrient medium is 20 mg/l to about 20 g/l. The concentration of arginine is maintained in an amount of at least 20 mg/l or at least 100 mg/l or at least 300 mg/l or at least 500 mg/l or at least 1 g/l or at least 2 g/l or at least 3 g /l or at least 4 g/l or at least 5 g/l or at least 10 g/l or at least 20 g/l. The arginine concentration can be maintained between 20 mg/l and approximately 20 g/l or between 100 mg/l and 20 g/l or between 500 mg/l and 20 g/l or between 500 mg/l and 10 g/l or between 1 g/l and 10 g/l or between 5 g/l and 10 g/l or between 5 g/l and 20 g/l. Arginine can be provided to the culture such that the consumption of arginine by the culture is at least 20 mg arginine per gram dry cell weight or at least 100 mg arginine per gram dry cell weight or at least 1 gram arginine. arginine per gram of dry cell weight or at least 5 grams of arginine per gram of dry cell weight or at least 10 grams of arginine per gram of dry cell weight. Arginine can be provided to the culture such that the consumption of arginine by the culture is between 20 mg and 20 grams per gram of dry cell weight or between 100 mg and 20 grams per gram of dry cell weight or between 1 gram and 10 grams per gram dry cell weight. The culture can consume at least 0.012 g of arginine to produce 1 g of biomass. The cellular requirement of arginine for biomass synthesis can be between 0.012 g per gram of biomass and about 24 g per gram of biomass. The arginine requirement for biomass synthesis can be from at least 0.012 g per gram of biomass or at least 0.024 g per gram of biomass, to about 0.048 g per gram of biomass or at least 0.120 g. per gram of biomass or at least 0.24 g per gram of biomass or at least 0.48 g per gram of biomass or at least 1.2 g per gram of biomass or at least 2.4 g per gram of biomass or at least 4.8 g per gram of biomass or at least 12 g per gram of biomass.

In some cases, increasing the concentration of arginine in the liquid nutrient medium to 2 to 80 times (or more) above the cellular requirement increases the doubling time of the microorganism by at least 10% or at least 20% or at least 30% or at least 40% or at least 50% or at least 60% or at least 70%.

When arginine was increased above the cellular requirement of Clostridium autoethanogenum , the production of acetate by the microorganism was reduced. Without wishing to be bound by theory, the inventors believe that the ability of Clostridium autoethanogenum to utilize arginine to produce ATP for growth negates the microorganism's need to produce acetate to acquire ATP when arginine is supplied.

All acetogenic microorganisms are described to produce acetate (Drake, Acetogenic Prokaryotes, in The Prokaryotes, 3rd edition, pages 354-420, New York, Ny, Springer, 2006), since acetate production provides the microorganism with an option to directly generate ATP from substrate-level phosphorylation via Pta (phosphotransacetylase) and Ack (phosphotransacetylase-acetate kinase). In particular, on a commercial scale, it is undesirable for microorganisms to produce acetate (or other organic acids required for the CoA transferase reaction) as a by-product, since acetate diverts carbon from target products and thus affects the efficacy and performance of the target products. Additionally, acetate may be toxic to microorganisms and/or may serve as a substrate for the growth of contaminating microorganisms. Furthermore, the presence of acetate makes it more difficult to recover and separate the target products and to control the fermentation conditions to favor the production of the target products.

Provision of arginine in excess of cellular requirements to a culture of Clostridium autoethanogenum results in greatly reduced acetate production compared to a culture where arginine is not provided in excess of cellular requirements. Also, the inventors have shown that the production of acetate increases in a culture, once the source of arginine has been completely depleted. Providing excess arginine to microbial culture can reduce acetate production by 20% or 30% or 40% or 50% or 60% or 70% or 80% .

The inventors have shown that arginine is stoichiometrically converted to ornithine, implicating the arginine deiminase pathway as the mechanism for arginine catabolism. This pathway would convert arginine to ornithine, ammonia, ATP, and CO2. Enhanced growth would be facilitated by supplying ATP and ammonium from arginine degradation. A method for improving the viability of a fermentation process is disclosed, wherein arginine is provided to the microbial culture in the absence of an alternative source of nitrogen.

It is well known in the art that the microorganism needs nitrogen for its growth. Normally, nitrogen is provided to the crop in the form of ammonium salt or ammonium hydroxide. Ammonia is normally produced by the Haber process, which is characterized by the following reaction:

N2(g) 3H2(g) =5=^ 2NH3(g) AH'0' = -92 kJ mol'1

Today, ammonia is produced primarily from natural gas and coal. In a normal process of producing ammonia from natural gas, hydrogen is obtained from natural gas and nitrogen comes from the atmosphere. Natural gas produces greenhouse gases, while ammonia itself does not produce greenhouse gases, ammonia production does. It is desirable to find and use sources of ammonia that are completely renewable. [http://www.agmrc.org/renewable_energy/renewable_energy/ammonia-as-atransportation-fuel/]. Ammonium hydroxide costs (28.0 to 30.0 p/p) are in the range of $9,600 per 1,000 kilograms.

Arginine (L-Arginine) was first isolated from the extract of lupine seedlings in 1886. It was later identified to be widely distributed in food and feed. Arginine can be produced by a variety of methods, including protein hydrolysis, chemical synthesis, and microbiological synthesis. Most of the L-arginine is produced by direct fermentation using renewable carbon sources.

[jn.nutrition.org/content/134/10/2854S.full]

The pathway identified from Example 3 allows for the conversion of 1 mole of arginine to 3 moles of ammonia. Therefore, arginine can provide an alternative source of nitrogen for bacteria, bringing ammonia directly into metabolism, while still providing the benefits described above. Since 1 molecule of arginine can be broken down into 3 molecules of ammonia, the lower amounts needed lead to significant savings due to the lower price of arginine (99% food grade arginine costs around $17-18,000/1000). kg, while 30% industrial grade ammonia costs ~10 to $11,000/1000kg Sigma Aldrich or Fisher) and reduced handling. Furthermore, arginine can be sustainably obtained from biological sources, for example by fermentation (T. Utagawa, J. Nutr., 2004, 134, 2854S-2857).

The arginine deiminase pathway proceeds through three enzymatic steps, catalyzed by arginine deiminase (EC 3.5.3.6), a carbamoyltransferase (ornithine carbamoyltransferase, putrescine carbamoyltransferase) (EC 2.1.3.3), and a carbamate kinase (EC 2.7.2.2). . Respective enzymes have been identified in the genome of C. autoethanogenum.

A genome-engineered C1-binding bacterium comprising an improved arginine deiminase pathway is disclosed. The engineered C1-binding bacterium may comprise one or more enzymes selected from the group consisting of: arginine deiminase (Ec 3.5.3.6), carbamoyltransferase (ornithine carbamoyltransferase, putrescine carbamoyltransferase) (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2 ), where each enzyme is an overexpressed endogenous enzyme, an endogenous enzyme mutated from an exogenous enzyme. The C1-binding bacterium may be a Clostridium bacterium. The bacterium may be Clostridium autoethanogenum.

While not wishing to be bound by theory, the inventors believe that to increase the yield, in particular, if accumulation of intermediates such as citrulline, of the pathway is observed, the respective genes may be over-expressed or genes from other sources may be introduced. and express heterologously by one skilled in the art using the methods described above (WO2012/053905, WO2012/115527 and WO2013-180584).

A method for producing at least one product from a substrate is disclosed, the method comprising culturing a genome-engineered C1-fixing bacterium comprising one or more enzymes selected from the group consisting of: arginine deiminase (EC 3.5.3.6), carbamoyltransferase ( ornithine carbamoyltransferase, putrescine carbamoyltransferase) (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2), where each enzyme is an overexpressed endogenous enzyme, an endogenous enzyme mutated from an exogenous enzyme.

A genome-engineered C1-fixing bacterium is disclosed which comprises one or more enzymes selected from the group consisting of ornithine racemase (EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12) and 2-amino-4-oxopentanoate thiolase. Without wishing to be bound by theory, the inventors believe that the respective genes can be overexpressed or genes from other sources can be introduced and expressed heterologously by one skilled in the art using the methods described above (WO2012/053905, WO2012/115527 and WO2013-180584). The body can also adapt and evolve to use ornithine more efficiently if it adapts to grow with arginine over time. This is supported by the observation of an accumulation of ornithine.

The inventors have identified an arginine repressor that controls gene expression by binding to a palindromic operator sequence that is located approximately 45 bp upstream of the arginine deiminase start codon. The addition of arginine causes the repressor to dissociate from the operator sequence, allowing transcription of genes downstream of the operator sequence. A genome-engineered recombinant bacterium comprising at least one heterologous gene is disclosed, said heterologous gene being provided downstream of an argR-binding operator sequence, wherein gene expression of the at least one heterologous gene can be activated by the addition of arginine.

A method for producing at least one product is disclosed, the method comprising providing a carbon source to a culture containing a genome-engineered bacterium comprising at least one heterologous gene, said heterologous gene being provided downstream of an argR binding operator sequence, provide the culture with arginine and ferment the culture. The at least one heterologous gene may be a heterologous gene in the biosynthetic pathway of the product.

In one example, the heterologous genes may encode a metabolic pathway that requires ATP for product synthesis. For example, the mevalonate pathway is a heterologous pathway that converts acetyl-CoA to isopentenyl diphosphate at a cost of 3 moles of ATP per mole of isopentenyl diphosphate. Using the method described, the expression of the heterologous mevalonate pathway could be activated by the addition of arginine, which would also provide ATP for the mevalonate pathway through arginine degradation via the arginine deiminase pathway.

In another aspect, arginine can be utilized through an arginine decarboxylation pathway. Without wishing to be bound by theory, the inventors believe that arginine can be decarboxylated to agmatine and CO2 by the enzyme arginine decarboxylase. Agmatine can be further converted to N-carbamoyl putrescine by the enzyme agmatine deiminase, also producing ammonium. Putrescine carbamoyltransferase can convert N-carbamoyl-putrescine plus phosphate to putrescine plus carbamoyl-phosphate. Carbamoyl phosphate plus ADP is converted to ammonium ATP CO2 by carbamate kinase through the same mechanism as the arginine deiminase pathway. The net yield of ammonia and ATP is the same as the arginine deiminase pathway but with two different intermediates (agmatine and N-carbamoyl putrescine) and a different byproduct (putrescine). The Putrescine is a higher value by-product than ornithine or arginine and can be used as a starting material for the production of a variety of polymers, including nylon-4,6 (Qian et al. 2009, Biotechnol. Bioeng. 104, pp 651-662) and polyurethane.

The bacterium may be modified to overexpress one or more endogenous genes, express one or more mutated endogenous genes, or heterologously express one or more exogenous genes, encoding enzymes selected from the group consisting of ornithine racemase, ornithine aminomutase, p-subunit (OraE), ornithine aminomutase, a-subunit (OraS), 2,4-diaminopentanoate dehydrogenase, 2,4-diaminopentanoate dehydrogenase, 2-amino-4-oxopentanoate thiolase, p-subunit, 2-amino-4-oxopentanoate thiolase, a-subunit, and functionally equivalent variants of the same. Alternatively, the bacterium can be modified to express one or more exogenous genes selected from the group consisting of ornithine racemase, ornithine aminomutase, p-subunit (OraE), ornithine aminomutase, a-subunit (OraS), 2,4-diaminopentanoate dehydrogenase, 2,4-diaminopentanoate dehydrogenase, 2-amino-4-oxopentanoate thiolase, p-subunit, and 2-amino-4-oxopentanoate thiolase, a-subunit. Endogenous genes may come from Clostridium sticklandii (Figure 23). Without wishing to be bound by theory, the inventors believe that overexpression of these genes will result in increased yields in the arginine deiminase pathway, particularly when ornithine accumulation is observed in the parent strain. Alternatively, it is believed that the bacterium may adapt and evolve to use ornithine more efficiently. The bacterium can be selected to grow on arginine. The bacterium may comprise one or more genetic modifications that disrupt an arginine:ornithine transporter. Genetic modification can disrupt the expression of CAETHG 3023 and CAETHG_3024 (Genbank GeneID: 17336441 and 17336442; Genbank Protein Registry Number: YP_008700482.1 and YP_008700483.1). Preferably, the genetic modification is a knockout mutation of the gene. Inactivation of the arginine:ornithine transporter results in decreased export of ornithine from the bacterial cell and allows metabolism of ornithine by the cell. Additionally, the bacterium can be modified to express an alternative arginine transporter to import arginine without exporting ornithine. Alternatively, the bacterium can be modified to express an ornithine importer to allow recovery of excreted ornithine. Each of these approaches will increase the bacteria's ability to metabolize ornithine.

Example 1 shows the production of alanine from arginine by C. autoethanogenum. A method for the production of one or more alanine-derived products, including alanine-derived products 3-hydroxypropionate (3-HP) or acrylic acid, is disclosed. Acrylic acid is an important product with uses in polymeric flocculants, dispersants, coatings, paints, adhesives, and binders for leather, paper, and textiles with an estimated global demand of 5 million tons in 2014. 3-HP is a platform for acid acrylic, methyl acrylic acid, acrylamide or 1,3-propanediol.

A genome-engineered C1-fixing bacterium is disclosed comprising at least one heterologous enzyme selected from the group consisting of: enzymes for converting alanine to malonyl-semialdehyde and 3-HP, enzymes for converting alanine to alanyl-CoA, enzymes for converting 3-HP into acrylyl-CoA, enzymes to convert alanyl-CoA to acrylyl-CoA, and enzymes to convert acrylyl-CoA to acrylate.

The C1-binding microorganism may be an acetogenic carboxydotrophic microorganism. Examples of suitable C1-fixing microorganisms include Clostridium, Moorella, Oxobacter, Peptostreptococcus, Acetobacterium, Eubacterium, or Butyribacterium. The microorganism can be selected from the group of microorganisms identified in Table 5.

examples

Example 4 further illustrates the invention but should of course not be construed as limiting its scope in any way.

Table 1: PETC-MES media without yeast extract

continuation

T l 2: Frml i n min i n n nr i n finl n l

Example 1

Identification of preferred amino acids of acetogen C. autoethanogenum and enhanced growth with arginine supplementation

This example demonstrates that arginine, histidine, aspartate, and glutamate are clearly preferred over other amino acids and are consumed in >80-fold higher yields than are required for biomass synthesis by the C. autoethanogenum acetogen DSM10061 and complementation. of arginine in defined medium allows C. autoethanogenum to grow with ~3 hr tD, which is ~3-fold faster compared to yeast extract (YE) supplementation.

Clostridium autoethanogenum DSM10061 was obtained from DSMZ (The German Collection of Microorganisms and Cell Cultures, InhoffenstraBe 7 B, 38124 Braunschweig, Germany).

PETC-MES medium without YE was supplemented with various amino acid formulations: 20AA, 7AA, 8AA, 14AA, 12AA, 4AA and Arginine (Table 2), to identify if any amino acid is supporting growth. Solutions were filter sterilized and made anoxic by sparging with N2.

First, a 20AA medium was designed based on the amino acid composition of Clostridium acetobutylicum biomass (Lee et al. 2008) with final amino acid concentrations that theoretically support the production of 1 g PCS/l biomass.

Growth studies were carried out in batch cultures in 50 mL volume of medium in 125 mL nitrogen headspace serum bottles with shaking (unless otherwise noted) at 37°C. Optical density (OD) was measured at 600nm with a reading of ~0.5 outside the anaerobic chamber against fully oxidized medium as reference.

Organic acids, fructose and amino acids analyzed by HPLC. Analysis Organic acids, carbohydrates and alcohols were quantified by ion exclusion chromatography with an Agilent 1200 HPLC system and an Agilent Hiplex H column (300 *7.7 mm, PL1170-6830) with precolumn (SecurityGuard Carbo-H, Phenomenex PN: AJO-4490). In general, sugars and alcohols were monitored with a refractive index detector (Agilent RID, G1362A) set to positive polarity and optical unit temperature of 40 °C, while organic acids are monitored at 210 nm (Agilent RID, G1362A). MWD, G1365B) and/or with a refractive index detector. 30 µl of sample was injected onto the column using an autosampler (Agilent HiPALS, G1367B) and the column temperature was maintained at 65°C using a thermostatted column compartment (Agilent TCC, G1316A). The analytes were eluted isocratically with H2SO44 mM at 0.6 mL/min for 26 min. Fructose, sucrose and glucose were analyzed separately at a column temperature of 15 °C and using high purity water (18.2 MQ-cm) as mobile phase and eluted isocratically at 0.4 ml/cm. min for 21 min. Chromatograms were integrated using ChemStation (Dietmair S., Timmins N. E., Gray P. P., Nielsen L. K., Kromer J. O.: Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol. Analytical biochemistry 2010, 404:155-164). Amino acids were measured and quantified as previously described (R. B. McQualter, C. Bellasio, L. K. Gebbie, L. A. Petrasovits, R. W. Palfreyman, M. P. Hodson, M. R. Plan, D. M. Blackman, S. M. Brumbley, and L. K. Nielsen, Plant Biotechnol. J., 2015) .

Although the maximum OD achieved in the 20AA medium was slightly lower (~20%) than in the yeast extract, equally rapid growth (doubling time tD = ~9 h; growth rate p = 0.077) was observed at baseline. of sampling (Figure 1).

AA analysis surprisingly showed a preferential and rapid utilization of aspartate, glutamate, serine, histidine and threonine AAs (FIG. 2). Arginine was rapidly depleted from the media. More importantly, serine and threonine were consumed >6-fold and aspartate, glutamate, histidine and arginine >20-fold more than the yields needed for biomass production, indicating their use for purposes other than incorporation into biomass. Remarkably, alanine accumulation, rather than consumption, was observed in both media during growth. Based on these results, subsequent media with 2- and 4-fold higher concentrations were designed for minimal media, faster growth, and higher OD: 14AA media by omitting six AAs that were either not consumed at all or they slowly and medium consumed 8AA by further reducing the amino acids.

In 14AA medium with increased 4*AA concentrations, a higher maximum OD was achieved compared to growth in yeast extract (Figure 3). Notably, the maximum OD achieved in the 8AA medium matched that of the 20AA medium, showing that a more minimal AA formulation can support good growth. Increasing AA concentrations had a positive effect on doubling time: faster and equally fast growth was achieved in 14AA and 8Aa media, respectively, compared to yeast extract and 20AA medium (Figure 4). ).

AA analysis confirmed the previous findings and further highlighted the importance of the AAs aspartate, glutamate, histidine, and arginine (Figure 5 and Figure 6). Notably, arginine was the most preferred amino acid as it was already depleted at OD=0.24. Among the other AAs, serine and threonine were used the fastest. Again, a significant accumulation of alanine was observed in both media. Interestingly, ornithine was also produced. Aspartate, glutamate, histidine, and arginine were consumed in yields >10 times higher than those required for biomass production, indicating their use for energy generation.

Based on these results, two subsequent media were designed to obtain faster growth: a 12AA medium that omitted glutamine and tryptophan from the 14AA medium and increased serine, threonine, and cysteine concentrations 8-fold, and a medium consisting of only in the 4 Aa identified aspartate, glutamate, histidine and arginine at 80 times compared to the 20AA medium (2 and 20 times compared to the 14AA medium); the 4AA medium containing only the "top 4 AAs" aspartate, glutamate, histidine, and arginine at concentrations 80 times higher compared to the 20AA medium.

The 4AA medium design allowed a higher maximum OD than the 12AA medium and matched the maximum OD obtained in the yeast extract and the 20AA medium (Figure 7). In both 4AA and 12AA media, rapid growth was measured - tD ~2.5 h - up to OD ~0.3, after which growth slowed. It is important to note that the ~2.5 hr tD achieved is ~4 times faster compared to supplementation with 1 g/L yeast extract as commonly used.

The initial rapid growth followed by a slowdown can be explained by arginine depletion. From the AA analysis data of the 12AA medium, it can be seen that arginine is the preferred AA and was completely depleted at OD = 0.37 (FIG. 8). These data also indicate that arginine strongly enhances growth along with glutamate, aspartate, and histidine by supporting tD ~2.5 h as measured between OD 0.1 and 0.3 while growth slowed after . that arginine was depleted between OD 0.3 and 0.37. Simultaneous accumulation of ornithine was observed. Again a significant accumulation of alanine was detected.

Growth deceleration after arginine depletion is also evident in 4AA medium (FIG. 9). In addition to ornithine, citrulline accumulation was also detected during arginine catabolism. In addition to alanine accumulation, small amounts (~0.3 mM) of lysine and valine accumulated for the first time, while also an unknown maximum (8.3 min retention time) showed areas of increasing maxima.

Since the initial rapid growth was unexpected, only a couple of samples could be used to calculate the tD. Therefore, a BugLab biomass monitoring device was attached to a serum bottle in 4AA medium to continuously monitor the biomass increase before the medium was depleted of arginine. This experiment confirmed the rapid growth in 4AA before arginine depletion, as the tD was calculated to be ~2h (FIG. 10).

Even supplementation with arginine alone resulted in very good growth with equally high maximum OD than in 4AA medium and yeast extract. Growth of C. autoethanogenum DSM10061 in PETC-MES medium without YE with 5 g arginine/l 5 g fructose/l resulted in rapid initial growth early on (approximate tD 3 h) followed by slower growth in the second stage after arginine depletion (tD~40 h) (FIG. 11).

Surprisingly, during the initial phase of rapid growth, little acetate is produced. Acetate production is again linked to the growth of the slower growing second phase (Figure 12).

Figure 13 shows the rapid utilization of arginine. In addition to alanine accumulation, small amounts (~0.5 mM) of lysine and valine were produced similarly to the 4AA medium. The same unknown maximum (8.3 min retention time) observed in the 4AA medium showed areas of increasing maxima after arginine depletion.

To obtain a more accurate value of tD in Arginine medium, a BugLab experiment was performed to continuously monitor biomass increase before arginine depletion. This experiment confirmed rapid growth before arginine depletion, as the tD was calculated to be ~3 h (FIG. 14). Faster growth on Arginine compared to yeast extract is shown in Figure 15.

Example 2

Enhanced autotrophic growth with arginine supplementation

This example demonstrates a higher specific growth rate and lower acetate production for autotrophically growing Clostridium autoethanogenum acetogen DSM23693 when supplemented with arginine.

Clostridium autoethanogenum DSM23693 (a derivative of Clostridium autoethanogenum DSM10061 ; US Patent 2013/0217096) was obtained from ^d S ^m Z (The German Collection of Microorganisms and Cell Cultures, InhoffenstraBe 7 B, 38124 Braunschweig, Germany).

Growth was carried out in PETC-MES medium without yeast extract (Table 1) using standard anaerobic techniques (Hungate, Meth Microbiol, 3B: 117-132, 1969; Wolfe, Adv Microb Physiol, 6: 107-146, 1971 ) and 151.68 kPa (22 psi) headspace pressure of the CO/CO2/H2 gas mixture (composition: 50% CO, 20% CO2, 2% H2, 28% N2).

To study the effect of arginine supplementation, 5 g/l arginine was added to the medium and culture growth and metabolite production during autotrophic growth were compared with the control without arginine.

Growth was followed by optical density measurement with a Thermo Genesys 20 spectrophotometer. Acetate (acetic acid), ethanol, 2,3-butanediol or lactic acid metabolites were measured by high performance liquid chromatography (HPLC) on an LC Agilent with refractive index (RI) detection at 35°C. Samples were prepared by diluting 400 μl with 100 μl of 5-sulfosalicylic acid solution (1% w/v in 1M sulfuric acid), followed by 3 min centrifugation at 14,000 rpm; the supernatant was transferred to a glass bottle for analysis. The separation was carried out with a 10 j l injection on an Alltech IOA-2000 column (150 mm x 6.5 mm *8 Jim) at 0.7 ml/min and 65 °C under isocratic conditions, using a mobile phase. of 5 mM sulfuric acid.

The experiment was carried out in two biological replicates with cultures in triplicate (n = 3) for each condition in a volume of 40 ml of medium in 1 L Schott bottles at 37 °C with orbital shaking (120 rpm, orbital shaking). In each case, the acetogenic C. autoethanogenum strain DSM23693 was pre-cultured in PETC-MES without yeast extract to an OD 600 nm of 0.3 and a single preculture was used for inoculation.

In the first experiment at the following conditions: PETC-MES without yeast extract syngas at 151.68 kPa (22 psi) (initial OD600nm = 0.005) and PETC-MES without yeast extract syngas at 151.68 kPa (22 psi) 5 g arginine/L (initial OD 600 nm = 0.005).

In the repeat experiment, a culture was also inoculated in medium supplemented with arginine but without the addition of CO/CO2/H2 gas (100% N2 as headspace). This experiment comprised the following three conditions: PETC-MES without yeast extract syngas at 151.68 kPa (22 psi) (initial OD600nm = 0.03), PETC-MES without yeast extract syngas at 151, 68 kPa (22 psi) 5 g arginine/L (initial OD 600 nm = 0.003) and PETC-MES without yeast extract 5 g arginine/L (initial OD 600 nm = 0.003). Media containing arginine were inoculated at a lower initial density than media without arginine to accommodate the surprisingly increased specific growth rate with arginine observed in the first experiment.

In both experiments, the arginine cultures grew surprisingly fast (4 doublings in <15 h corresponding to a doubling time of tD = 3.5 h and a specific growth rate j = 0.198) to an OD600 nm “0.8 , while the growth of the control was much slower, with a doubling time tD = 7.3 h and a specific growth rate j = 0.095 (Figures 16 and 17). Arginine supplementation decreased the doubling time and increased the culture-specific growth rate during autotrophic growth, thus by more than 50%.

A log-transformed plot of autotrophic growth of C. autoethanogenum clearly demonstrates the decrease in arginine complementation doubling time (FIG. 18). The calculated doubling times are tD = 7.3 hrs ± 0.2 for autotrophic growth without arginine supplementation and tD = 3.5 hrs ± 0.2 with arginine supplementation, a 52% decrease.

With and without arginine, the cultures reached the same final density during autotrophic growth, indicating that arginine was not used as a carbon source but only served to increase the specific growth rate (Figures 16 and 17). This was also confirmed in the culture where no gas was supplied, no growth was observed within 48 hours in cultures supplemented with arginine but without the CO/CO2/H2 gas mixture (FIG. 17). This supports the hypothesis that arginine is not used as a carbon source under these conditions, but instead CO/CO2 is used as a carbon source and ATP is supplied by arginine metabolism.

Autotrophic growth of acetogens is usually related to acetate (acetic acid) production, as acetate formation generates ATP through substrate-level phosphorylation in the acetate kinase reaction that is essential for growth. As such, all acetogens isolated to date were found to produce acetate. However, acetate formation is undesirable from a process perspective as it diverts carbon away from target products and is known to be toxic to microorganisms already at low concentrations of a few percent (J. Ballongue, E. Masion , J. Amine, H. Petitdemange and R. Gay, Appl. Microbiol. Biotechnol., 1987, 26, 568-573; G. Wang and DI Wang, Appl. Environ. Microbiol., 1984, 47, 294-8) . In the growth medium supplemented with 5 g arginine/l, surprisingly, no net acetate production was observed until the pause in growth at OD 600 nm ≤ 0.8 only at this stage was acetate produced (FIG. 19). The results of this set of experiments suggest that C. autoethanogenum can use arginine to produce ATP for growth and therefore does not need to produce acetate when arginine is supplied.

Example 3

Identification and optimization of arginine utilization pathways

This example demonstrates how arginine provides additional ATP for the cell and can fuel central metabolism of acetogens.

As demonstrated in Example 1, arginine converts stoichiometrically to ornithine. I also know Citrulline accumulation was observed at some time points. Without wishing to be bound by theory, this observation implicates the arginine deiminase pathway as the mechanism.

This pathway would convert arginine to ornithine, ammonia, ATP, and CO2. Enhanced growth would be facilitated by supplying ATP and ammonium from arginine degradation. This supply of ATP also eliminates the need for acetogens to make acetate.

The arginine deiminase pathway proceeds through three enzymatic steps, catalyzed by arginine deiminase (EC 3.5.3.6), ornithine carbamoyltransferase (putrescine carbamoyltransferase) (EC 2.1.3.3), and a carbamate kinase (EC 2.7.2.2). Respective enzymes (including two arginine/ornithine transporter genes/enzymes) have been identified in the genome of C. autoethanogenum, ornithine carbamoyltransferase and carbamate kinase are present in multiple copies (Table 4). Enzymes that can catalyze the same reactions are also present in other organisms, including many acetogens including C. Ijungdahlii, C. scatologenes, C. drakei, and Acetonema longum (Table 5). This pathway is also present in C.stricklandii or E. coli.

Table 4: Identified genes/enzymes of the ar inine deiminase pathway in C. autoethano enum

Table 5: Identified genes/enzymes of the deiminase pathway in other organisms

continuation

continuation

(continuation)

To increase the yield, in particular, if accumulation of intermediates such as citrulline, of the pathway is observed, the respective genes become overexpressed or genes from other sources can be introduced and expressed heterologously by one skilled in the art using methods described above [US 2013/344547, US 2013/330809, US 2013/323820, US 2013/224838, US 2013/224839, US 2011/256600, US 2011/236941.

Ornithine itself can be further converted to alanine, which has been shown to accumulate in Example 1 as well. This conversion also generates additional reducing equivalents NAD ^p (H), as well as another ammonia molecule and the fundamental building block acetyl- CoA of CoA.

As shown in Figure 23, the conversion of ornithine to alanine and acetyl-CoA occurs through enzymatic steps ornithine racemase (EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase ( EC 1.4.1.12) and 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10). The respective enzymes have been described, for example, from Clostridium sticklandii or environmental samples and homologues have been identified in C. autoethanogenum (Table 6).

Table 6: Identified genes/enzymes of the ornithine to alanine and acetyl-CoA conversion pathway of c.

sticklandii or homologous environmental samples of C. autoethane enum.

continuation

To increase the yield of the pathway, particularly since ornithine accumulation was observed, the respective genes from C. autoethanogenum can be overexpressed or genes from C. sticklandii or environmental samples from Table 6 can be introduced and expressed heterologously. by a person skilled in the art using the methods described above (W2012/053905, WO2012/115527, WO2013/180584). The body can also adapt and evolve to use ornithine more efficiently if it adapts to grow with arginine over time.

To achieve flux in the pathway, it may also be necessary to inactivate the arginine:ornithine transporter (CAETHG_3023-24) to prevent ornithine from being exported out of the cell. Said inactivations can be achieved by one skilled in the art using the methods described above (W2012/053905, WO2012/115527, and WO2013/180584). In addition, it may be necessary to add an alternative arginine transporter. An alternative approach to inactivating the arginine:ornithine transporter could be to introduce an ornithine importer, so that ornithine can be further metabolized.

The identified pathways were also simulated in a genome-scale model reconstruction to show the effect of metabolism and confirm the identified pathways.

A genome-wide metabolic reconstruction for C. autoethanogenum was generated based on published methods (L.-E. Quek and LK Nielsen, Genome Inform., 2008, 21, 89-100; CG de Oliveira Dal'Molin, L. -E. Quek, RW Palfreyman, SM Brumbley and LK Nielsen, Plant Physiol., 2010, 152, 579-89, C. Licona-Cassani, E. Marcellin, L.-E. Quek, S. Jacob and LK Nielsen, Antonie Van Leeuwenhoek, 2012, 102, 493-502.). The genome-scale model core was reconstructed using the SEED model annotation line (CS Henry, M. DeJongh, AA Best, PM Frybarger, B. Linsay, and RL Stevens, Nat. Biotechnol., 2010, 28, 977- 82). The reconstruction retained all SEED model reaction attributes, including unique reactions, compound identifiers, and reaction reversibility. The model was manually completed in Excel (Microsoft Corporation) to facilitate annotation and comment, in particular, the central metabolism and arginine utilization pathways identified above were manually selected. From this gene-centric database, a 2D reaction-centric SBML (System Biology Markup Language, http://www.sbml.org) representation was generated using a Java application (Oracle Corporation). Constraint-based analysis and reconstruction were performed with the COBRA toolkit (http://opencobra.sourceforge.net/) (J. Schellenberger, R. Que, RMT Fleming, I. Thiele, JD Orth, AM Feist, DC Zielinski, A. Bordbar, NE Lewis, S. Rahmanian, J. Kang, DR Hyduke, and B. 0. Palsson, Nat. Protoc., 2011, 6, 1290-307). A set of scripts for constraint-based modeling that run within the MATLAB environment. A flux balance analysis (Orth et al., 2010) was used to predict essential nutrients for growth and a shadow price analysis was performed to investigate the beneficial effects of amino acids (AAs) on Clostridium autoethanogenum, and to determine which AA have the greatest impact on ATP production. Flux balance analysis (FBA) has been used to predict essential nutrients for growth (Fan et al., 2014; Song et al., 2008), the effects of supplementation with AA in the synthesis of the target product (Licona-Cassani et al., 2012) and in conjunction with shadow price analysis (Hillier and Lieberman, 2010) to determine the substrates that have the greatest effect on ATP production (Teusink et al . ., 2006).

Conventional shadow price analysis can be misleading with AAs. If the shadow price analysis is performed under autotrophic conditions (i.e. zero absorption), it will show the opportunity cost of AA synthesis, i.e. the resources that could be released if a given AA were supplied by the environment. The most expensive AA to synthesize is not necessarily the best substrate for ATP production. For example, there may not be a downgrade path available.

To overcome this problem, a compensated shadow price analysis was performed. Each of the 20 AAs was allowed a maximum flux of 1.4 mmol/g PCS/h while biomass yield was maximized. This flux is the maximum specific rate of fructose uptake observed from C. autoethanogenum DSM10061 during preliminary growth experiments in conventional PETC-MES medium (including yeast extract (YE)) and fructose. AAs without a downgrade path cannot use the maximum allowed flow and therefore will have zero shadow price. Shadow price analysis identified nine AA: glutamine, histidine (HIS), cysteine (CYS), threonine, aspartate (ASP), arginine (ARG), glycine, serine, and glutamate (GLU), out of the 20 conventional that C. autoethanogenum should prefer, as they lead to faster growth.

The AA model prediction was confirmed in a medium of 20AAs for eight of the nine predicted AAs (Figure 2), excluding only glycine. Remarkably, the uptake of ASP, GLU, HIS and ARG by biomass was more than 20 times greater than that required for biomass synthesis. AA uptake per gram of biomass was compared to the concentration expected in the biomass (the cell requirement), based on measurements taken on Clostridium acetobutylcium (Lee et al., 2008).

Surprisingly, in another engineered 12AA medium, uptake of ASP, GLU, HIS and ARG was found to be more than 120-fold higher than required for biomass synthesis and allowed for significantly faster growth. This indicates the possible participation of ASP, GLU, HIS and ARG in power generation. Interestingly, along with arginine consumption during the rapid growth phase, an accumulation of ornithine was detected (Figure 8), pointing towards the possible involvement of the arginine deiminase (ADI) pathway in ATP supply. addition to cells. It is noteworthy that the rapid growth observed initially (tD = 2.5 ± 0.1 h) is very close to the growth predicted with 20 AA (tD = 2.9 h).

Interestingly, substantially lower biomass production of acetate was observed in media containing ASP, GLU, HIS and ARG (4AA medium) (8.2 ± 0.2 mmol/g PCS) and medium containing only arginine (4AA medium). ARG) (6.7 ± 0.7 mmol/g PCS) during growth when arginine was abundant compared to YE (36.6 mmol/g PCS). Acetate production increased markedly after arginine depletion (46.5 ± 11.2 and 34.5 ± 9.3 mmol/g PCS for 4AA medium and ARG medium, respectively), showing that the ability to catabolize arginine greatly reduces the need for an acetogen to produce acetate.

Heterotrophic cultures of C. autoethanogenum grown in 12AA medium accumulated ornithine simultaneously with arginine consumption (Figure 8). The same was observed in bioreactor experiments with 4AA medium and arginine medium only with a high fraction (approximately 60%) of the consumed carbon excreted as ornithine (FIG. 22). Furthermore, a significant carbon flux to CO2 was detected and also a notable accumulation of citrulline. Other minor byproducts included acetate, alanine, and ethanol. Taken together, the significant carbon fluxes to ornithine, CO2, and citrulline suggest that arginine was catabolized via the ADI pathway, explaining its growth-enhancing effect via ATP supply (Figure 21). Furthermore, the complete stoichiometric conversion of arginine to ornithine, CO2, and citrulline implies that arginine was metabolized strictly for energy and not for biomass synthesis. This is consistent with the result that no growth was observed when only arginine was supplemented to Schott bottles containing PETC-MES without YE pressurized with N2 gas.

The involvement of the ADI pathway in facilitating faster growth was predicted by initial in silico simulations in 4AA medium and arginine alone. Total specific ATP production rate (qatp; mmol ATP/g PCS/h) was approximately 6-fold higher in the latter conditions (SIM 4 and 5) compared to calculations with fructose (SIM 1) as the sole carbon source. (Table 7). The same was observed when the model was constrained with experimentally determined rates of substrate uptake and product secretion (excluding citrulline and CO2) from bioreactor experiments in 4AA media (SIM 6 and 9) and arginine alone (SIM 12 and 15), with a qatp approximately 3 to 4 times higher than expected in fructose alone. The faster growth observed (tD~3 to 4 h) with AA compared to growth predicted without AA supplementation (tD~14 h) may be explained by a significantly higher energy output from substrate-level phosphorylation. during the catabolism of arginine (by the carbamate kinase of the ADI pathway) and indirectly through the generation of a proton motive force by the production and excretion of NH4+. The latter is also beneficial to reduce the costs of the bioprocess due to the lower need to neutralize the pH with the addition of NH4OH.

The rapid growth and low acetate synthesis achieved through arginine supplementation is relevant to the biotech industry, as decreased carbon flux to the undesired acetate byproduct and additional ATP production from alternative pathways is essential. to expand the product spectrum of acetogens.

Genome-scale models can be used to estimate intracellular metabolic flux patterns and calculate carbon, redox, and energy balances by constraining the model with experimentally measured data (Bordbar et al., 2014; Dash et al. , 2016; O'Brien et al., 2015). A genome-wide metabolic model of C. autoethanogenum similar to that described by Marcellin (Low carbon fuels and commodity chemicals from waste gases - Systematic approach to understand energy metabolism in a model acetogen, Green Chem, 2016) was used. Genome-scale model analysis is performed for heterotrophic experiments in AA medium and arginine-only medium by performing flux balance analysis calculations as described above. The model was further constrained by specific substrate uptake and product secretion rates (excluding citrulline and CO2) and cellular tD. Maximizing ATP dissipation (ie unaccounted for ATP costs; see below) was used as the target function to perform FBA (maintenance energy costs were not included here).

Rapid growth in 4AA medium and ARG medium was facilitated by approximately 3 to 4-fold higher qATP (29.3 ± 5.5 and 19.9 ± 1.0 mmol ATP/g PCS/h for 4AA medium and ARG medium). A ^r G, respectively (SIM 6 and 9, and SIM 12 and 15 in Table 7) compared to the value of 6.8 mmol ATP/g PCS/h predicted for growth with fructose alone (SIM 1). The inventors consider that the faster growth observed in 4AA medium compared to ARG medium is explained by the 2-fold higher specific flux through the ATP-producing acetate kinase reaction (Figure 24). both media ~53% ATP was produced via the ADI pathway (Figure 24) showing that arginine catabolism completely reorganized energy metabolism Interestingly, although arginine uptake is favored in many bacteria ( Abdelal, 1979; Adamberg et al., 2006; Lahtvee et al., 2011; Mehmeti et al., 2011), his contribution to the production energy supply is variable.

The predicted optimal flux pattern during heterotrophic growth in fructose and AA was also analyzed. When constraining the model with experimentally measured substrate uptake rates and previously calculated unaccounted for ATP costs, and maximizing biomass yield, these calculations predicted faster growth (mean 4AA tD = 1.4 ± 0.2 h, SIM 7 and 10; mean ARG tD = 2.2 ± 0.1 h, S ⁱ M 13 and 16) compared to experimentally observed (tD = 2.8 ± 0.1 h and tD = 3.7 ± 0.0 h, respectively). Furthermore, in the simulations, ornithine produced during arginine catabolism was catabolized to GLU, demonstrating that it is possible to further metabolize ornithine via the ornithine pathway described above. Therefore, the fastest predicted growth comes from the catabolism of GLU to pyruvate using the methylaspartate pathway (as noted above) and its subsequent conversion to acetyl-CoA and acetate resulting in reduced Fd and ATP.

Example 4

Growth on arginine as sole nitrogen source

This example demonstrates the replacement of ammonium as the nitrogen source with arginine.

Nitrogen is an essential nutrient for bacteria, and ammonium is commonly used in fermentations and is also used as a nitrogen source in published media formulations for acetogens (M. Kopke, C. Held, S. Hujer, H. Liesegang A Wiezer A Wollherr A Ehrenreich W Liebl G Gottschalk P Dürre Proc Natl Acad Sci U.S. A. 2010 107 13087-92 J Mock Y Zheng A. P. Mueller, S. Ly, L. Tran, S. Segovia, S. Nagaraju, M. Kopke, P. Dürre, and R. K. Thauer, J. Bacteriol., 2015, 197, 2965-2980;H. Richter, M. E. Martin and L. T. Angenent, Energies, 2013, 6, 3987-4000 J. L. Cotter, M. S. Chinn, and A. M. Grunden, Bioprocess Biosyst Eng., 2009, 32, 369-80 M. Straub, M. Demler, D. Weuster -Botz and P. Dürre, J. Biotechnol., 2014.). However, ammonium production is dependent on abundant energy supplies, predominantly natural gas or liquefied petroleum gases (LPG) via the Haber-Bosch process (www.essentialchemicalindustry.org/chemicals/ammoinia.html), and thus , is not a sustainable source. In addition, ammonium ions influence the pH of the fermentation.

The pathway identified from Example 3 allows for the conversion of 1 mole of arginine to 3 moles of ammonia. Therefore, arginine can provide an alternative source of nitrogen for bacteria, bringing ammonia directly into metabolism, while still providing the benefits described above. Since 1 molecule of arginine can be broken down into 3 molecules of ammonia, the lower amounts needed lead to significant savings due to the lower price of arginine (99% food grade arginine costs around $17-18,000/1000). kg, while 30% industrial grade ammonia is ~$10 to $11,000/1000 kg from Sigma Aldrich or Fisher) and with reduced handling. Furthermore, arginine can be sustainably obtained from biological sources, for example by fermentation (T. Utagawa, J. Nutr., 2004, 134, 2854S-2857).

To demonstrate that arginine can be used as the sole nitrogen source for the DSM23693 Clostridium autoethanogenum acetogen, the organism is grown in YE-free PETC-MES medium omitting 1 g/L ammonium chloride (NH4Cl) with 0.33 g /l of non-synthetic L-arginine monohydrochloride (Sigma Aldrich; A6969).

Example 5

Arginine supplementation leads to increased production of unnatural products from heterologous pathways, in particular, ATP uptake pathways

This example demonstrates that arginine supplementation increases the production of non-natural products from heterologous pathways.

The production of various non-natural products in acetogens from gas has been demonstrated [W2012053905, WO2012115527, WO2013180584, WO2013185123, WO2013191567]. Some level of acetate is usually seen as a by-product, as the body generates ATP from substrate-level phosphorylation via the acetate kinase reaction. This is particularly the case for pathways that require ATP, such as, but not limited to, pathways for the production of isoprene or other terpenoids (mevalonate pathway), or the production of products from fatty acids such as biodiesel (via fatty acid biosynthesis). However, it is also the case for other fermentation pathways that do not directly require ATP but also do not produce the same amount of ATP per acetyl-CoA as acetate formation, for example, but not limited to, the production of isopropanol, acetone, butanol (via the ABE route).

Isopropanol production from gas was simulated using flow balance analysis (FBA) with the C. autoethanogenum genome-scale model (described in example 3). Heterologous routes (Table 4) were added to the model and simulations were performed on the maximum theoretical yield with and without arginine supplementation (Table 5).

Table 8: Heterologous Routes

Table 9: FBA analysis of maximum theoretical yields of isoprene or isopropanol target products from 2H2 n in m l m n r inin n mm l P h:

The simulation shows that arginine supplementation can significantly increase the maximum theoretical production yields of the target compounds isopropanol and isoprene in both CO and CO2/H2. The maximum production of isopropanol from CO could be significantly increased by more than 20% from 5.06 mmol/g PS/h to 6.42 mmol/g PS/h already with small amounts of arginine uptake (2 mM, 0.34 g/l). For the mevalonate pathway that consumes ATP for the production of isoprene from CO, the maximum production could be increased even more significantly by more than 30% from 1.97 mmol/g PS/h to 2.58 mmol/g PS /h. The effect is even more pronounced with CO2/H2, where the maximum production of isopropanol can be increased by more than 25% from 5.0 mmol/g PS/h to 6.36 mmol/g PS/h and the production of isoprene by a 45% from 1.26 mmol/g PS/hr to 1.83 mmol/g PS/hr. This clearly demonstrates that arginine can be used to drive the production of unnatural target molecules.

The simulation also shows that when 2 mM arginine is used together, production is even increased with less CO uptake, demonstrating that arginine supplementation improves the efficiency of carbon in a target molecule.

Example 6

Use of Arginine Repressor as a Genetic Switch to Drive Expression of Heterologous Pathways This example demonstrates that arginine can be used as a genetic switch to drive expression of heterologous pathways.

All genes in the identified arginine deiminase pathway of example 3 were found to be clustered and an arginine repressor ArgR was also found. Operator sequences (DNA sequences to which the argR protein binds to prevent gene transcription) have been identified in a variety of microorganisms including E. coli (Tian et al. 1992, J. Mol. Biol, 226, pgs. 387-397) and the binding site is generally conserved across bacterial lineages (Makarova et al. 2001, Genome Biol. 2, pp 1-8).

The arginine repressor controls gene expression by binding to a palindromic operator sequence that is approximately 45 bp upstream of the arginine deiminase start codon. The adition of arginine causes the repressor to dissociate from the operator sequence, allowing transcription of genes downstream of the operator sequence. In a prescient example, heterologous gene expression can be activated by the addition of arginine. Heterologous gene expression will be repressed by the argR protein if the argR binding operator sequence is added to the upstream region of the heterologous genes. Subsequently, gene expression can be activated by the addition of arginine.

In one example, the heterologous genes may encode a metabolic pathway that requires ATP for product synthesis. For example, the mevalonate pathway is a heterologous pathway that converts acetyl-CoA to isopentenyl diphosphate at a cost of 3 moles of ATP per mole of isopentenyl diphosphate. Using the method described, the expression of the heterologous mevalonate pathway could be activated by the addition of arginine, which would also provide ATP for the mevalonate pathway through arginine degradation via the arginine deiminase pathway.

Example 7

Optimization of the efficiency of the joint utilization of arginine with gaseous substrates of CO and/or H 2 and/or CO 2

To achieve efficient co-utilization of arginine with gaseous substrates of CO and/or H2 and/or CO2, it may be necessary to deregulate. This could be achieved by inactivating the arginine repressor ArgR described above to remove repression of the arginine deiminase pathway genes. Such inactivations can be achieved by someone skilled in the art using methods described previously [W2012053905, WO2012115527, WO2013180584]. However, removal of the arginine repressor does not allow heterologous gene expression to be activated by the addition of arginine as described above. An alternative approach would be to remove the previously described operator binding sequences upstream of the arginine deiminase pathway operon or to replace the region upstream of the arginine deiminase pathway operon that includes the operator sequence and promoter sequence with a constitutive or synthetic promoter. Said modifications can be achieved by someone skilled in the art using methods described previously [W2012053905, WO2012115527, WO2013180584]. Suitable constitutive and synthetic promoters are, for example, but not limited to, the Pfdx ferredoxin promoter, Ppta acetate kinase promoter, or Ptet or PIPL12 which have been described previously [US 20160160223; Nagaraju et al., Genome editing of Clostridium autoethanogenum using CRISPR/Cas9, Biotechnol Biofuels. 2016; 9: 219].

Similarly, the promoter regions of genes responsible for the utilization of CO and/or H2 and/or CO2, such as the Wood-Ljungdahl cluster or the Hyt operon (Brown et al. Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia. Biotechnology for Biofuels 2014 7:40) can be replaced with constitutive and synthetic promoters or the respective inactivated or attenuated regulators. Those skilled in the art will be able to identify such regulators from transcriptomic data as described in Example 10.

Example 8

Alternative pathway of arginine utilization and putrescine production

This example demonstrates an alternative pathway of arginine utilization that can generate putrescine as a by-product.

Another possible route for arginine utilization is through arginine decarboxylation rather than deamination: arginine can be decarboxylated to agmatine and CO2 by the enzyme arginine decarboxylase. Agmatine can be further converted to N-carbamoyl putrescine by the enzyme agmatine deiminase, also producing ammonium. Putrescine carbamoyltransferase can convert N-carbamoyl-putrescine plus phosphate to putrescine plus carbamoyl-phosphate. Carbamoyl phosphate plus ADP is converted to ammonium ATP CO2 by carbamate kinase through the same mechanism as the arginine deiminase pathway. The net yield of ammonia and ATP is the same as the arginine deiminase pathway but with two different intermediates (agmatine and N-carbamoyl putrescine instead of citrulline) and a different byproduct (putrescine instead of ornithine). Putrescine is a higher value by-product than ornithine or arginine and can be used as a starting material in the production of a variety of polymers, including nylon-4,6 (Qian et al. 2009, Biotechnol. Bioeng. 104, pp. 651-662) and polyurethane (Dahiyat et al. 1993, J. Biomater. Sci. Polym. Ed., 4, pp. 529-543).

Table 10.

Example 9

Alanine production and conversion to 3-HP or acrylic acid via heterologous pathways

This example demonstrates the conversion of alanine to 3-hydroxypropionate or acrylic acid.

In example 1 the production of alanine from arginine by C. autoethanogenum has been demonstrated. Alanine can be further converted to high value products 3-hydroxypropionate (3-HP) or acrylic acid.

The conversion of alanine to acrylate has been demonstrated in Clostridium propionicum (Dalal RK, Akedo M., Cooney CL, Sinskey AJ (1980) A microbial route for acrylic acid production. Biosources Dig 2:89-97). The conversion can be done by malonyl-semialdehyde and 3-HP, or by alanyl-CoA. Both 3-HP and alanyl-CoA can be converted to acrylyl-CoA and then to acrylate. Genes/enzymes isolated from C. propionicum can be introduced and heterologously expressed by one skilled in the art using previously described methods to produce 3-hydroxypropionate or acylate [W2012053905, WO2012115527, WO2013180584].

Example 10

Transcriptomic analysis

Transcriptome analysis of duplicate bioreactor cultures of C. autoethanogenum growing heterotrophically in 4AA medium (see Table 2 for details) was performed with RNA sequencing. During the balanced growth phase and uptake of all 4AA, approximately 35 mL of 0.3 g PCS/L culture was collected, pelleted (5000* g for 3 min at 4 °C) and resuspended in 5 mL of . RNAlater (Qiagen). The sample was stored at 4 °C overnight, centrifuged (4000* g for 10 min at 4 °C), and the pellet was stored at -80 °C until further processing. Frozen pellets were thawed, total RNA extracted, and mRNA libraries prepared as described in Marcellin et al., 2013. Sequencing was performed with an Illumina Hiseq-2000 sequencer.

Gene expression patterns in 4AA medium were compared with RNA-seq data from the same C. autoethanogenum strain (DSM10061) grown heterotrophically in conventional PETC-MES (including YE) and previously published (Marcellin et al., 2016). Sequencing reads from both heterotrophic conditions were trimmed to avoid misreads and then aligned/realigned to the genome using TopHat2 (Kim et al., 2013) with two mismatches allowed per read alignment. Transcript abundances were estimated using the FPKM function of Cufflinks using upper quartile normalization. CuffDiff was used to estimate differentially expressed transcripts and Cuffnorm for data normalization. A q value of less than 0.05 (using the false find rate; Benjamini and Hochberg, 1995) was used to determine significant changes in gene expression.

RNA sequencing (RNA-seq) analysis of the transcriptome was performed to obtain further confirmatory evidence for the involvement of the ADI pathway at the level of gene expression. To do this, samples were taken from duplicate bioreactor biological cultures of C. autoethanogenum growing heterotrophically in 4AA medium during the balanced growth phase. This RNA-seq dataset was compared with the previously published dataset (Marcellin et al., 2016) of the same C. autoethanogenum strain (DSM10061) grown heterotrophically (fructose) in the conventional PETC-MES medium. which contains YES.

Transcriptome analysis demonstrated the role of the ADI pathway, as all ADI pathway genes were upregulated by more than 500-fold (q-values of <0.001) when cells were cultured in PETC-MES supplemented with 4AA compared to YE). In addition, more than 380-fold upregulation (q values of <0.001) of putative arginine-ornithine antiporter genes (CAETHG_3023 and 3024) was observed. Of the four genes associated with the degradation of citrulline to carbamoylphosphate and ornithine in C. autoethanogenum, the present data indicate that ornithine carbamoyltransferase (CAETHG_3022) is the efflux catalytic protein (645-fold upregulation; q < 0.001) during catabolism of ARG in C. autoethanogenum since its isoenzymes were down-regulated (CAETHG_0449 and 0591) or did not show changes (CAETHG_2082) between the compared conditions. Similarly, of the five genes associated with carbamoyl phosphate degradation to CO2, the carbamate kinase of CAETHG_3025 appears to be the efflux catalyst protein (623-fold loop regulation; q < 0.001) as its FPKM value is 1000 times higher than the FPKM values of its isoenzymes. Example 11

Optimizing the efficiency of arginine incorporation in central metabolism

The transcriptomic analysis identified genes that in the natural metabolism determine the flow of utilization and incorporation of arginine in central metabolism as ornithine carbamoyltransferase (CAETHG_3022) and carbamate kinase (CAETHG 3025). To improve efficiency, these genes can be overexpressed using strong or inducible promoters, for example, but not limited to, the Pfdx ferredoxin promoter, Ppta acetate kinase promoter, or Ptet or PIPL12 which have been described previously [US 20160160223; Nagaraju et al., Genome editing of Clostridium autoethanogenum using CRISPR/Cas9, Biotechnol Biofuels. 2016; 9: 219].

The use of the terms "a", "an" and "the" and similar references in the context of the description of the invention (especially in the context of the following claims) are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The expressions "comprising(n)", "having(n)", "including(n)", and "containing(n)" are to be interpreted as open-ended expressions (i.e. meaning "including (n), but not limited to"), unless otherwise indicated. The enumeration of ranges of values herein is merely intended to serve as a shorthand method of individually referring to each separate value that falls within the range, except as otherwise noted herein and each separate value is incorporated herein. the specification as if individually listed herein.

All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples or illustrative language (e.g., "such as") provided herein is merely for the purpose of further illustrating the invention and does not pose a limitation on the scope of the invention, unless otherwise noted. claim otherwise. No language in the specification is to be construed as indicating that anything not claimed is essential to the practice of the invention.

Preferred embodiments of this invention are described herein. Variations of these preferred embodiments may be apparent to those skilled in the art from reading the foregoing description. The inventors hope that such variations will be used by those skilled in the art as appropriate and the inventors intend that the invention be practiced other than as specifically described herein. Still further, any combination of the elements described above in all possible variations thereof is encompassed by the invention unless otherwise indicated or clearly contradicted by the context.

Claims (11)

1. A method for culturing a C1-fixing microorganism, comprising flowing a gaseous substrate comprising one or more of CO, CO2 and H2 into a bioreactor containing a culture of a C1-fixing microorganism in a liquid nutrient medium; where arginine is provided to the crop as the sole source of nitrogen; further, wherein the C1-binding microorganism is a recombinant genetically modified microorganism comprising an arginine metabolism pathway; and further wherein the C1-binding microorganism is Clostridium autoethanogenum, Clostridium Ijungdahlii or Clostridium ragsdalei. 2. The method of claim 1, wherein the arginine metabolism pathway comprises an arginine deiminase pathway and/or an arginine decarboxylase pathway, wherein the arginine deiminase pathway comprises one or more enzymes selected from group consisting of arginine deiminase (EC 3.5.3.6), ornithine carbamoyltransferase (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2), and wherein the arginine decarboxylase pathway comprises one or more enzymes selected from the group consisting into arginine decarboxylase (EC 4.1.1.19), agmatine deiminase (EC 3.5.3.12), putrescine carbamoyltransferase (EC 2.1.3.6), and carbamate kinase (EC 2.7.2.2). 3. The method of claim 2, wherein the arginine deiminase pathway comprises one or more of an overexpressed endogenous enzyme, a mutant endogenous enzyme, or an exogenous enzyme. 4. The method of claim 1, wherein the concentration of arginine in the bioreactor is at least 300 mg/l. 5. The method of claim 1, wherein the culture does not produce acetate. 6. The method of claim 1, wherein the arginine is catabolized by the arginine metabolism pathway to produce ammonia, wherein the C1-fixing microorganism uses ammonia as a nitrogen source. 7. The method of claim 1, wherein the C1-binding microorganism comprises one or more genetic modifications in the arginine metabolism pathway, wherein the one or more genetic modifications are selected from the group consisting of (i) a switch mutation of regulatory elements and (ii) a replacement of operator binding sites or natural promoters by constitutive or synthetic promoters. 8. The method of claim 7, wherein the disruptive mutation is an inactivation of the arginine repressor argR. The method of claim 7, wherein the replacement is a replacement of an arginine deaminase pathway operon promoter with a constitutive or synthetic promoter. 10. The method of claim 1, wherein the C1-binding microorganism comprises a switch mutation in an arginine:ornithine transporter. 11. The method of claim 1, wherein the C1-binding microorganism is Clostridium autoethanogenum LZ1561, which is deposited under DMSZ accession number DSM23693.